In this paper, we present the ImmunoDisk, a fully automated sample-to-answer centrifugal microfluidic cartridge, integrating a heterogeneous, wash-free, magnetic- and fluorescent bead-based immunoassay (bound-free phase detection immunoassay/BFPD-IA). The BFPD-IA allows the implementation of a simple fluidic structure, where the assay incubation, bead separation and detection are performed in the same chamber. The system was characterized using a C-reactive protein (CRP) competitive immunoassay. A parametric investigation on air drying of protein-coupled beads for pre-storage at room temperature is presented. The key parameters were buffer composition, drying temperature and duration. A protocol for drying two different types of protein-coupled beads with the same temperature and duration using different drying buffers is presented. The sample-to-answer workflow was demonstrated measuring CRP in 5 µL of human serum, without prior dilution, utilizing only one incubation step, in 20 min turnaround time, in the clinically relevant concentration range of 15–115 mg/L. A reproducibility assessment over three disk batches revealed an average signal coefficient of variation (CV) of 5.8 ± 1.3%. A CRP certified reference material was used for method verification with a concentration CV of 8.6%. Our results encourage future testing of the CRP-ImmunoDisk in clinical studies and its point-of-care implementation in many diagnostic applications. Immunoassays can specifically detect protein biomarkers in various sample matrices such as serum, urine or saliva via the binding of an antigen by antibodies ,2,3. TheCentrifugal microfluidic systems are suitable candidates for the shifting of diagnostic practice from centralized to PoC settings. Their closed cartridges can support fully automated analyses, do not require external pumps for fluid handling because they utilize changes in frequency and temperature to run their protocols, and are less prone to bubble clogging ,14. Pre-One main structural feature of most of the demonstrated immunoassays based on centrifugal systems is that they use magnetic or fluorescent micro/nanoparticles  as assay components requires solutions for their pre-storage directly on the cartridge in order to achieve fully automated sample-to-answer analysis at the PoC . The preIn the context of the state of the art, we present a fully integrated centrifugal microfluidic system, the ImmunoDisk, implementing, for the first time, an immunoassay method that combines a bead-based and wash-free configuration without additional surface treatments or density media, with all reagents pre-stored, and using bound-free phase detection (BFPD). The bound-free phase detection immunoassay (BFPD-IA) is a heterogeneous assay based on ELISA principles and utilizes magnetic and fluorescent beads, the former acting as capture phase and the latter as the detection agent . The siThe sample-to-answer ImmunoDisk performance in terms of robustness and reproducibility was assessed by detecting C-reactive protein (CRP) in human serum and by using a certified reference material (CRM). CRP was selected for demonstration due to its broad use as an inflammation biomarker  to suppoOverall, our parametric study on the pre-storage of protein-coupled beads, in combination with the simplified, wash-free microfluidic integration of the BFPD-IA workflow, can open up new perspectives on the integration of heterogeneous, bead-based immunoassays within  microfluidic systems for user-friendly, rapid, sample-to-answer immunoassay-based diagnostics.The novel BFPD immunoassay was presented and evaluated in detail in a previous publication by Johannsen et al. . The folIn order to assess the activity of the dried bead-coupled antibodies and antigens after pre-storage and resuspension, we used a negative control of the CRP assay in a microtiter plate, as described in a previous publication . This neThe coupling of anti-human CRP antibodies , Waltham, MA, USA) onto the tosyl-activated surface of the magnetic beads was described in detail in a previous publication by Johannsen et al. . In shorThe coupling of native CRP protein  onto the carboxyl-activated surfaces of the fluorescent beads , 0.2 µm, Thermo Fisher Scientific, Waltham, MA, USA) was described in detail in a previous publication by Johannsen et al. . In shorFor the evaluation of the drying buffer as part of the parametric study, 5 µL of drying buffer containing either the magnetic or the fluorescent beads was dried in a microtiter plate  in an incubator  at 37 °C for 17 h and 6 h for the magnetic and fluorescent beads, respectively. They were stored in the dark in a container with silica beads. The beads were resuspended for 5 min at 750 rpm  before the CRP assay was conducted see . The dryThe following additives to PBS were used (in different combinations) to test different drying buffer compositions for drying the anti-CRP antibody-coupled magnetic beads: (1) trehalose , (2) sucrose , (3) BSA, (4) Polyethylene glycol 1000 (PEG1000) , (5) Tween80  and (6) CHAPS . Additionally, two buffers from the companies Merck KGaA  and GSK , which they use for the lyophilization of their proteins, were tested. The detailed composition of the additives in these buffers is given by Mensink et al.  (we dissThe following additives to PBS were used (in different combinations) to test different drying buffer compositions for drying the CRP antigen-coupled fluorescent beads: sucrose, trehalose, PEG1000 and Tween80.w/v) trehalose and 50% (w/v) PEG1000) to give a final bead concentration of 20 µg/µL. The fluorescent beads with native CRP antigen on their surfaces were diluted directly in their drying buffer containing PBS with 10% (w/v) trehalose and 10% (w/v) sucrose to give a final bead concentration of 3.8 µg/µL. Then, 5 µL of each of the magnetic and the fluorescent bead solutions was pipetted into dedicated storage chambers on the disk and then dried in an incubator at 39 °C for 1 h. The disks were stored at RT in a container with silica beads  before sealing. The assay buffer  was stored in stickpacks  and simulated utilizing a network simulation processed with MATLAB Simulink Simscape R2016a  that was improved for the development of centrifugal microfluidic disks, as described in detail by Schwarz et al. . The dist et al. ) that arA centrifugal microfluidic disk, the ImmunoDisk, was developed for the automation of the BFPD-IA workflow with all reagents pre-stored A,B. The The disk is mounted into the processing device  pipetting the sample into the inlet or pipetting the liquid solutions of fluorescent and magnetic beads prior to drying, (ii) metering the assay buffer, (iii) pumping the assay buffer radially inwards into chamber #3 and (iv) valving and transferring the mixture  from chamber #4 into the multipurpose chamber #5.The performance of the metering process and the volume transfer of the pneumatic pumping and the pneumatic valving steps were evaluated by pipetting 5 µL of assay buffer into the sample inlet and 110 µL of assay buffer into the stickpack chamber. The fluidic protocol was started and the volume that was transferred into the incubation chamber was measured (N = 6) with an average of 78.3 ± 0.5 µL. This corresponds to a deviation of 4% from the targeted 75 µL and shows a high reproducibility, with a coefficient of variation (CV) below 1%. To assess the reproducibility of the resuspension of the fluorescent beads and the fluidic valving, fluorescent beads were dried in their assigned storage chamber #4 before 75 µL of buffer was pipetted into the inlet to ensure a controlled volume. The buffer was transferred to chamber #4 containing the fluorescent beads, which were resuspended, mixed and transferred to the multipurpose chamber. After batch-mode mixing for 200 s, the fluorescence signal was measured (N = 9). The average signal of 1132 ± 30 RFU showed a CV of only 2.7%. Part of these  variances may be attributed to the manual pipetting and variances of the detector. We expect even lower variances for automatically produced disks in the future. The combination of pneumatic and temperature-induced overpressure during valving ensures that all of the resuspended fluorescent beads are transferred to the multipurpose chamber. All these results demonstrate low variation deriving from the microfluidic operations and show that the disk and its fluidic protocol are well-suited for performing the BFPD-IA.The multipurpose chamber was designed with some special features to facilitate the bead and liquid handling and to optimize the various assay steps, all in a single chamber A. (i) A A key element of the BFPD-IA method is the detection in the unbound phase, which consists of only the fluorescent beads, after the bound phase (the complexes of magnetic beads with fluorescent beads and/or CRP antigen from the sample) has been removed. Due to its special features, and in combination with the appropriate centrifugal protocols, the multipurpose chamber allows this key element of the BFPD-IA to be transferred to the disk cartridge by integrating the single-step assay incubation (including mixing), the separation of the bound phase (sedimentation) and the detection in the same chamber. This is enabled by the fact that, based on the design of the assay itself, the unbound fluorescent beads are not sedimented, while the bound phase is. The distance traveled by a bead at specific frequencies can be estimated using Stokes’ law modified for centrifugal forces ,60 , because the former can be conducted with dispensing robotics and large ovens, while the latter would require a pick-and-place robot and strict environmental humidity conditions.The requirements for the pre-storage on the ImmunoDisk  were (i) maintaining the functionality of the proteins, (ii) the fast and complete resuspension of the beads and (iii) the prevention of aggregate formation. Additional manufacturing-related requirements demand that there should be (iv) stable bead pellets and, ideally, (v) no additional treatment of the (cartridge) surfaces.The parameters that may influence the storage result are the drying buffer (including protein stabilizing additives), the drying temperature and the drying duration next to the fixed parameters of bead type, proteins and volumes. Thus, we conducted a detailed experimental investigation of these parameters. In the first step, we tested a non-exhaustive list of candidate drying buffers, temperatures and durations and evaluated each of them by means of assay performance and pellet resuspension. We also took into account that the final drying conditions should be compatible with both bead types (magnetic and fluorescent), as well as both biomolecule types coupled on the beads’ surface .This part of our work should serve as an orientation for the reader in the field of pre-storage of protein-coupled beads. We make no claim of a complete investigation of the drying parameters and conditions, nor was the goal to examine the impact of these conditions on the structure/function of the proteins and beads.We started our study by investigating suitable drying buffers first for the antibody-coupled magnetic beads (MB), while keeping the antigen-coated fluorescent beads (FB) in solution, and then for the antigen-coupled fluorescent beads, while keeping the antibody-coated magnetic beads in solution. We used the BFPD-IA, where all reagents were in the liquid state, as a reference. The signal intensity resulting from each assay performed with a specific drying buffer was subtracted from the reference and this ΔIntensity was used to assess the functionality of the reagents after drying . A higher CV could indicate the formation of aggregates or unreproducible resuspension properties and therefore, the CV should be comparable to the CV of the reference assays. Images of dried beads were taken after drying with the attempt to find a correlation between the visual properties of the pellet and the post-rehydration assay performance, but we did not find any. Therefore, we considered only the assay performance itself as an evaluation criterion for the drying process. These evaluation experiments were first performed on a microtiter plate, and the best-performing drying buffers were then tested on the cartridge.Representative additives were selected from classes of reagents that have been reported to support the stabilization of proteins, such as sugars, polymers and detergents ,61, or tDifferent drying buffer combinations were tested for the air drying of MB, and the most representative results are shown in w/v) for either of the two disaccharides.Two different disaccharides were tested, trehalose and sucrose. Disaccharides are non-reducing sugars and, in contrast to reducing sugars, do not denature proteins with a Maillard reaction . It has w/v), we had quite low ΔIntensity values, almost as low as in the case of sucrose (w/v) PEG1000 combined with 10% (w/v) trehalose or 10% (w/v) sucrose was not sufficient to improve the results, which were ~3× worse when using the sugars alone (w/v) and combined it with 10% (w/v) sucrose or 10% (w/v) trehalose, the ΔIntensity was drastically reduced, although with some large error bars for the case of 10% (w/v) sucrose/25% (w/v) PEG1000 (w/v) (w/v) sucrose vs. 10% (w/v) trehalose, We also examined a representative polymer additive, PEG1000. PEG as a protein stabilizer has been controversially discussed in the literature and is often said to be used with caution, as its effect is protein-dependent ,67,68. W sucrose A, which rs alone . When we PEG1000 , which w00 (w/v) B. This cw/v) trehalose and 25% (w/v) sucrose: BSA, which might help to reduce non-specific binding to surfaces during drying [v/v) Tween80 to trehalose reduced the ΔIntensity substantially, although the CV was increased.Three further additives were combined, in different concentrations, with both 25%  sucrose, 25% (w/v) sucrose with 0.1% (w/v) CHAPS, 10% (w/v) sucrose with 50% (w/v) PEG1000, and 10% (w/v) trehalose with 50% (w/v) PEG1000 had similar ΔIntensity results. As mentioned earlier, the selection of the drying buffer should be based not only on the degree of stabilization of protein function (assessed through the assay performance), but also on the quality of the resuspension of the dried magnetic bead pellet. The corresponding photo of the well after resuspension of the dried MB is shown in We also tested commercial buffers from the companies Merck and GSK  that werw/v) trehalose with 50% (w/v) PEG1000 in PBS. It showed superior resuspension quality and reproducibility in the cartridge, as was the case in the microtiter plate  trehalose combined with 10% (w/v) sucrose in PBS gave the lowest ΔIntensity value, and therefore, this drying buffer was selected for the fluorescent beads.Due to the above, we decided to continue with the drying buffer for FB containing only sugars. The fact that different drying buffers had to be selected for the antibody-coated magnetic beads and the antigen-coated fluorescent beads might be due to the different nature of the proteins, which we also observed with the different impact of PEG1000 on the drying results. The drying buffer for the CRP antibody-coupled magnetic beads also showed promising results when used for magnetic beads that were coupled with matrix metalloproteinase 9 (MMP-9) antibodies  measuredOur experimental observations indicate that for different types of proteins, it is necessary to test different additives in different concentrations, and that there is no universal solution for all proteins.Having defined the biochemistry of the drying buffers for the magnetic and fluorescent beads, the next step in our parametric study was to define the drying conditions. Unlike the buffers, which do not have to be the same, the duration and temperature must be the same for both drying buffers, as the two bead solutions must be dried simultaneously and in the same cartridge.We used a design of experiments (DoE) approach with Minitab  to evaluate the influence of temperature and duration on the drying of the protein-coupled beads. The DoE showed that the combination of drying temperature and duration has the strongest influence on the result, along with the drying duration alone. The drying temperature by itself has no significant influence on the result. It should be kept in mind that the DoE tested low and high temperatures of 37 °C and 45 °C and that higher temperatures can have a strong influence, for example, if the proteins are denatured. For more information on the DoE please see The drying duration was set to 1 h, which is favorable from a disk manufacturing perspective. A screening of different drying temperatures, shown in Having specified the drying buffers and conditions, we briefly explored the short-term stability (two weeks) of the dried beads in a disk cartridge at RT. We first used dried magnetic beads and liquid fluorescent beads. We compared the average results on day 1 and day 16 using the negative control. In this configuration, we observed that the signal on day 16 after drying differed by 1.7% compared to that on day 1. In a subsequent set of experiments, we used dried magnetic beads as well as dried fluorescent beads. As above, we used the negative control. The negative control was measured after 1, 4, 11 and 15 days of storage at room temperature. In this case, the average signal on day 15 after drying differed by 4.7% compared to day 1. The measurements over the four different days after storage show a variation of 6.3% (N = 7). These are promising preliminary results and the deviations are within the inter-assay variation. The storage stability over time needs to be evaluated further with longer storage durations and over all concentrations covered by the calibration curve, but this would go beyond the scope of this work.In a final step, the results from the developments in the areas of reagent pre-storage and assay transfer to microfluidics were demonstrated in a sample-to-answer configuration on the disk with all reagents stored at RT. CRP was used as a target antigen and CRP-free human serum as a complex matrix.A calibration curve was obtained by measuring CRP-spiked human serum standards using three batches of disks on three different days A. This aThe obtained calibration curve  the BFPD-IA on a microfluidic cartridge. Notably, the initial version of the BFPD-IA, conducted in a microtiter plate, included a step to transfer the bound-free phase to a separate well for detection after the separation step. However, through the interplay between microfluidic design and protocols, we managed to not only reduce the manual handling steps to just one (insertion of the sample), but also to omit the transfer of the bound-free phase and to achieve incubation, separation and detection all in the multipurpose chamber. Thus, the few and easy steps of the BFPD-IA itself , togetheFor the full integration of a bead-based immunoassay PoC system like the one we propose, it is inevitable that the pre-storage of protein-coupled beads comes into focus. The parametric study of this topic presented in this publication revealed some interesting findings.We show for the first time, to the best of our knowledge, the combination of a polymeric and a disaccharide as additives for the pre-storage of protein-coupled beads using air drying. We observed that the buffer with a combination of PEG1000 and trehalose performed best for the CRP antibody-coated magnetic beads, but that PEG itself proved to be incompatible with the CRP antigen-coated fluorescent beads, most probably due to the different nature of the coupled protein. The common drying protocol for the two different types of beads and proteins on the same disk is also an important outcome. This can open the way for other bead-based assays to be integrated in microfluidic systems.In its fully integrated version, the ImmunoDisk is able to measure high concentrations of CRP in a 5 µL human serum sample, which is inserted directly into the cartridge with no prior dilution steps and no washing steps on disk. These conditions simplify the fluidic integration and reduce the footprint on disk. Importantly, the ImmunoDisk was processed on a device, the LabDisk Player 1 functional model, which was developed for the automation of nucleic acid amplification technologies  and for which different applications have already been shown (e.g., respiratory tract infections , tropicaIn this work, we show, for the first time, the full sample-to-answer integration and automation of a heterogeneous wash-free, bead-based, bound-free phase detection immunoassay for the detection of CRP in serum in the clinically relevant range, including in situ detection and pre-storage of all involved reagents. The results show the great potential of the ImmunoDisk to be processed on the same centrifugal microfluidic processing device that performs PCR or isothermal amplification.For the pre-storage of the protein-coupled beads, we investigated all the key parameters for the air drying of proteins on particles and gained some key insights on the influence of additives and the importance of the drying temperature and duration combination. Thus, this work contributes significantly to the research methodology around the pre-storage of beads coupled with proteins, for which there is, to the best of our knowledge, limited literature available, unlike for proteins or beads alone. From the microfluidic perspective on the pre-storage of protein-coupled beads, we propose using a storage chamber on the cartridge for a more reproducible and robust storage and release of the protein-coupled beads. Furthermore, a multipurpose chamber is introduced that facilitates the assay incubation, separation and detection, leading to a simplified, robust, space-saving microfluidic design. The future outlook for this work includes the expansion of the pre-storage protocols to other protein assays, covering applications such as oral health, cardiovascular diseases or sepsis, and the implementation of the CRP-ImmunoDisk with clinical samples and potentially in combination with molecular diagnostics for improved patient management at the PoC.


The cytokine interleukin 6 (IL-6) is involved in the pathogenesis of different inflammatory diseases, including cancer, and its monitoring could help diagnosis, prognosis of relapse-free survival and recurrence. Here, we report an innovative microfluidic approach that uses the fluidization of magnetic beads to specifically extract, preconcentrate and fluorescently detect IL-6 directly on-chip. We assess how the physical properties of the beads can be tuned to improve assay performance by enhancing mass transport, reduce non-specific binding and multiply the detection signal threefold by transitioning between packed and fluidization states. With the integration of a full ELISA protocol in a single microfluidic chamber, we show a twofold reduction in LOD compared to conventional methods along with a large dynamic range (10 pg/mL to 2 ng/mL). We additionally demonstrate its application to IL-6 detection in undiluted serum samples. The increased interest for biomarkers has been accompanied by the emergence of a wide range of bioanalytical developments such as mass spectrometry or high throughput screening.Biomarkers are considered as objective quantizers of biological processes and particularly pathophysiological processes; they can be used for patient diagnosis or prognosis as well as to monitor disease progression or patient response to treatment. Biomarkers provide guidance through the development of new medicines5 through infections6 and cancer7. Cytokines are small proteins involved in cell signalling often used as indicators for disease monitoring8 such as in tumor progression9, liver diseases11 or hepatic inflammations and fibrosis12. In particular, interleukin 6 (IL-6) is involved in the response of the human immune system to infection and cellular injury14, being secreted by T cells and macrophages into the serum in case of acute and chronic inflammation. Recently, it has been suggested that coronaviruses may activate dysregulated host immune responses. Exploratory studies have suggested that interleukin-6 (IL-6) levels are elevated in cases of complicated COVID-1916. Thus, a quantitative analysis of cytokines in bodily fluids, and IL-6 in particular, can benefit the monitoring of a wide range of diseases. The current standard methods to detect and analyse cytokines are immunoassays, typically in the form of ELISA, microarrays and bead-based assays17. While immunoassays can be highly specific and sensitive, cytokine detection by batchwise immunoassay remains challenging due to their very low concentrations in biological samples down to sub pico or femto—molar concentration20.Among the different biomarkers , proteins have significantly demonstrated their potential and many of them are analysed and quantified for clinical diagnosis of diseases from asthma and allergies21, modified controlled-pore glass packet22 or carbon nanotube forests23. But while several microfluidic systems have already shown their efficiency for cytokine detection and quantification, there is still a need for new technologies and methods that can tackle the challenge of precise detection in complex matrices at low cost.The potential benefits of microfluidics are multi-fold: decrease analysis time, improve bioassays sensitivity, reduce sample and reagent volumes, decrease costs and miniaturize and integrate complex protocols. Impressive results of IL-6 detection in microfluidic systems have already been published relying on glass capillary27, initially as a miniaturization of conventional microtiter plate ELISA, the antibody being grafted at the surface of the microchannel29. To further improve the specific surface of interaction and consequently the surface to volume ratio, solid supports have been inserted in microdevices starting with mechanical trapping of micrometric polystyrene beads functionalized with antibodies30. The interest of using microbeads as solid support was exemplified by Teste et al. demonstrating theoretically and experimentally that the kinetics of target analyte capture is improved by using micro- and nano- magnetic particles compared to standard microtiter plates32. Since then, other strategies have been investigated leveraging electrokinetic and magnetic forces35, in particular in the form of droplet immunoassays combined with magnetics beads39. Previously, we developed the microfluidic magnetic fluidized bed, a beads-based microfluidic technology based on a homogeneous suspension of magnetic beads inside a microfluidic chamber40. A balance of drag and magnetic forces on the beads results in physical properties similar to those of a macroscale fluidized bed. The resulting high surface to volume ratio, constant mixing and compatibility with commercial and functionable beads make it attractive for bioanalysis integration. The porosity of the bed of beads plays a key role in the efficiency of the system, as it could affect the sample residence time and diffusion distances to the solid phase. This was demonstrated in a wide range of applications: bacteria analysis in raw samples42, detection of histone modifications43 and as a miniaturized platform for extra-corporeal circulation44. However, sensitive protein detection requires relatively complex multi-step protocols, challenging to integrate in a single device.Here we approach this challenge by improving current immunoassay-based protocols in an integrated bead-based microfluidic format. Immunoassays have been widely implemented in microfluidicHere we leverage the fluidized bed as a tool able to perform all the steps of an ELISA protocol for IL-6 detection: specific extraction, preconcentration, enzymatic binding and detection in a single microfluidic chip. We optimize and evaluate the performance of each step and study how the unique physical features of the mobile solid phase can be tuned to improve assay performance. We do this by adjusting the porosity of the system and the arrangement of the beads according to the molecular diffusion constant and the characteristics of the enzymatic reaction. Finally, we compare the results of our optimized system to the performance of current standard protocols for IL-6 quantitation.The washing buffer was prepared with Tris HCl , Bovine Serum Albumin “BSA”  and Tween 20 . The pH was adjusted at 7.5. The washing buffer was stored at − 4 °C.The enzymatic substrate MUP  was dissolved in the washing buffer at 10 mM, pH adjusted at 8.0. The substrate was kept at − 20 °C.Tosylactivated beads  were grafted following the Dynabeads datasheet with anti-human IL6 antibodies (Thermofisher Scientific from the kit CHC1263): they were shaken and incubated with anti-IL6 antibodies, Tris buffer and ammonium sulfate (3 M) at 37 °C for 18 h. Then, the beads were washed and resuspended in PBS with BSA 0.1% at a final concentration of 5 mg/mL. The detection antibodies and enzyme were provided by this same kit.45. It consists of an elbow channel leading to a diamond-shape chamber, with an opening angle of 13°. The height of the chamber and channel was set at 50 µm, and the total volume of the PDMS chamber was 0.6 µL.The microfluidic chip was described in previous publications46 was incubated inside the chip chamber for 2 h then rinsed with distilled water and dried with compressed air.Microfluidic chips were designed using a micro-milled mold. These molds were machined in brass pieces of 5 cm × 5 cm. The designs were a positive replica of the chip. The chips were fabricated by pouring polydimethylsiloxane  into the molds (concentration 1:10) and were bonded by oxygen plasma. A surface treatment of PDMA-AGE 0.5%The liquid flow was produced by a pressurization of the sample reservoir using a pressure controller  allowing to reach a range of pressures from 0.1 mbar to 1 bar, translated in flowrates between 0.1 and 3 µL/min. The outlet of the chip was connected to a flowrate controller , which allowed precise flowrate measurements and feedback control on the pressure based on the Maesflo software (Fluigent). Peek tubing  was used to connect the microfluidic chip to the other elements of the experimental set up. A tube (12 cm peek tubing of 0.063 mm diameter) was positioned at the entrance of the chip to increase the hydrodynamic resistance of the device. A permanent magnet made of NdFeB 1. 47 T  was aligned with the chamber axis at a 1.5 mm distance from the chip inlet.The sample (50 µL) and the detection antibodies  were incubated off-chip at room temperature for 50 min with continuous shaking. This mix (50 µL) was flowed inside the chip chamber containing functionalized magnetic beads at a 1 µL/min flowrate. The beads were then rinsed with the washing buffer for 30 min at 2 µL/min inside the chip prior to fluorescent detection.The sample (50 µL) was flown inside the chip chamber containing functionalized magnetic beads at 1 µL/min, then the detection antibodies  were flown at 1 µL/min. The beads were rinsed with the washing buffer for 30 min at 2 µL/min within the chip prior to fluorescent detection.The florescent signal was acquired on the outlet channel of the chamber, at half-height. For both methods, the signal of fluorescence was recorded for 6 min to reach a stable signal and the autofluorescence of the PDMS was subtracted to the signal.“Stop-and-go” reading process: The chamber was filled with 0.7 µL of MUP substrate at 10 mM at a flowrate of 400 nL/min. This step was followed by a 10-min incubation. The fluorescence signal was acquired while 6 µL of washing buffer  was flown through the beads at 1 µL/min.In-flow reading process: The signal was acquired as 6 µL of MUP at 10 mM was flown through the beads bed at 1 µL/min.The experiment in batch was performed in a 1.5 mL Eppendorf tube in which 25 µL of beads were introduced and washed three times in buffer. Beads were incubated for 100 min with the sample (10 ng/mL) or the buffer at room temperature (RT) under stirring, then washed three times. A second incubation was performed with the complex containing the detection antibody and enzyme for 100 min at RT under stirring followed by three washes. MUP substrate at 10 mM (6 µL) was added and incubated for 10 min at RT under stirring. The supernatant is then flown through the microfluidic chip to read the signal.It was performed with different concentrations of IL-6 cytokine (Thermofisher Scientific from the kit CHC1263) diluted in PBS and FBS to a final volume of 50 μL.The analytical characterization of the system was performed with n ≥ 2 and the optimization and calibration curves were obtained with up to 4 replicates for each value. The data were processed through Excel, as well as linear fitting and calculation of correlation coefficient. For Table 40. Beads are in free suspension in the liquid phase, a configuration that allows to avoid clogging issues. The microfluidic magnetic fluidized bed has shown interesting features regarding biomarkers analysis as it has already demonstrated its efficiency for nucleic acid analysis48, but so far it has not been applied to protein biomarker analysis in real samples. Here, we put forward the potential of this technology by combining both preconcentration in a dynamic configuration and on-beads detection. First, a high throughput extraction and preconcentration steps of the analyte are performed directly on the magnetic beads, then the detection of the target biomarker by sandwich ELISA is performed on chip and on beads in a very small volume with properties that can be tuned using the fluidized bed properties to achieve optimal performances.Microfluidic magnetic fluidized beds are based on the control of a homogenous suspension of magnetic beads in a microfluidic chamber. The drag force applied on the beads is due to the flow of liquid in the microchannel and is balanced by a magnetic force created by a permanent magnet45. These grafting strategies have been first compared in tube, using 20 µg of capture antibody per mg of beads for the tosyl-activated beads, and 4 µg of antibody per mg of beads for the MyOne Carboxylic beads as advised by the supplier, the capture antibody being here an anti-human IL6 antibody. To compare the different bead-grafting strategies, sandwich immunoassays were performed in the presence or absence of IL-6 to evaluate the specific to non-specific signal. Data shown in Table To implement a microfluidic fluidized bed-based bioassay for IL-6 detection, we first optimized the bead grafting with capture antibodies for IL-6 capture and preconcentration. In this approach, the magnetic beads in the fluidized bed have a pivotal role as they ensure an efficient extraction of the target in a continuous flow while being used in the second step as solid support to perform the sandwich ELISA. In order to optimize the bioassay performance, we first compared two widely used grafting strategies, based on covalently-grafted capture antibody using either tosyl-activated surface or carboxylic acid modified surfaces 3 , Tris EDTA , Tris HCl , and PBS . Our studies demonstrate a superiority of Tris–HCl buffer over the other ones (data not shown). In parallel, we have also investigated how the presence of BSA and Tween-20 affects the assay performances, both being well known to limit non-specific adsorption on beads as well as on PDMS. The presence of both additives indeed greatly improves the assay performances  and magnetic bead separation. But it has been recently demonstrated that single-step immunoassays can reach similar performances. In particular, Mai et al. reported a novel magnetic bead-based immunoassay in which the capture antibodies grafted onto magnetic beads and the detection antibodies can simultaneously bind to Aβ peptides in a single stepFor the off-chip immune complex formation, the sample, detection antibody and the enzyme were incubated together prior to their on-chip injection Figure .I. The eIn the case of the sequential injection, only the conjugation of the detection antibody by the enzyme was performed in tube prior to on-chip injection. Magnetic beads were functionalized as previously described Fig. I. Then, 50. In contrary, the nonspecific signal was significantly lower for sequential injection and the signal to noise ratio was two times higher for sequential injection compared to manual in-tube labelling  and 25 µg/mL (50 X). As shown in Fig. 40, but it has also been shown that the bead bed porosity can be tuned at will within the chip. A change of flowrate induces a change of the drag force applied to the beads. The balance between drag and magnetic forces is modified so that bed of beads expands as the flowrate increases. The influence of the flow rate on the on-chip immunoassay has thus been investigated as a change in porosity can impact not only the analysis time (from 25 to 100 min for 50 μL of sample) but also the diffusion distance, as well as the residence time of the target biomolecule within the bed of particles.With the conventional parameters of the bead-based immunoassay optimized, we next leveraged the fluidized bed format to improve assay performance. The microfluidic magnetic fluidized bed design has been optimized to reach a high homogeneity of bead distribution within the microchamber40, we know that the magnetic beads in microfluidic fluidized beds tend to self-organize in cylindrical clusters of diameter dc ≈12 μm due to bead-bead interactions. In a simple 1D approximation, we can consider the bed porosity ε being defined by the distance between these clusters ds and their size dc:From previous workd the time to reach a surface of capture by diffusion and tc the time to cross the magnetic bed by convection, we need to ensure that For an efficient capture, the analyte needs to be able to reach, by diffusion, a magnetic bead before leaving the fluidized bed due to the flow-driven convection. If we call td needed to travel the distance d = ds/2 allowing an analyte to reach the closer cluster by diffusion can be estimated with Einstein’s relation:The time t–8 cm2.s−151 and the aperture angle of the fluidized bed being α = 35° = 0.61 rad, we can estimate the ratio of times based on published measures of the bed length L and porosity ε40 at flowrates of 0.5, 1 and 2 μL/min  can then coexist in our system as a function of the flow rate applied and have been compared here to improve the last step of the immunoassay: the enzymatic reaction. The enzymatic reaction taking place within the fluidized bed offers the possibility to consider two detection modes with potentially improved performances. Indeed, after injection of the enzymatic substrate in the bed, the fluorescent signal can be detected either continuously (in flow method) or by sequentially changing the flowrate above and below the threshold (stop-and-go method).In the continuous in-flow detection approach, the enzymatic substrate was flowed continuously inside the chamber at 1 µL/min for 6 min Fig. A. The beIn the stop-and-go method, a given volume of enzymatic substrate (0.7 µL) is first flowed through the bed at 0.4 µL/min. The pressure is then decreased so that the substrate and the magnetic beads can be incubated in a packed bed regime for 10 min decreasing drastically the diffusion distances between the beads and the enzymatic substrate Fig. C. After As shown in Table To further evaluate the performances of the fluidized bed-based ELISA in terms of dynamic range and sensitivity, we established a calibration curve with the sequential injection protocol and a detection performed by stop-and-go mode for samples of 50 μL. This series of experiments was first performed with a wide range of IL-6 concentrations ranging from 10 pg/mL to 10 ng/mL in Tris HCl buffer 200 mM (at pH 7.5 with BSA at 1% and Tween 20 at 0.1%) as shown on Fig. 53 or to commercial immunoassays54 usually going from few pg/mL to ng/mL. Negative controls were performed with non-spiked buffer. We next evaluated the sensitivity of our newly developed immunoassay on the basis that significant signal is three standard deviation above the negative control. We obtained a limit of detection (LOD) at 6 pg/mL, more than two times lower than the one of the standard beads immunoassay (LOD is 15.6 pg/mL from manufacter’s data).A 50 µL sample was flowed through the chip at 1 µL/min then the detection antibody (0.5 µg/mL) was injected at room temperature for 50 min at the same flowrate. A quite large dynamic range was obtained with linear response from 10 pg/mL to 2 ng/mL, as shown in Fig. We finally applied the on-chip method to complex samples and performed a calibration curve with IL-6 spiked in fetal bovine serum (FBS) to validate our integrated approach towards complex sample matrix analysis Fig. C. This w57. Based on this first proof of concept, the modulable device may be further adapted for the detection of other cytokines.Altogether our results showed that the tunable properties of a magnetic and microfluidic fluidized bed allows to integrate an automated sequence of on-chip extraction and detection of Il-6. The fluidization regime can be used to limit the non-specific interactions and avoid clogging when working with complex matrices whereas the packed state could be used to enhance the detection step. The control of these two regimes (packed and fluidized states) allowed us to reach a relevant limit of detection in the tens of pg/mL, compatible with, for instance, the requirements of IL-6 detection in patient serum in sepsisMicrofluidic systems have demonstrated their potential to enhance bioassay performance and integration, particularly in the case of immunoassays. However, fully integrated multi-step protocols combining analyte extraction, preconcentration and detection in a single module are still challenging, particularly when addressing high sensitivity and/or compatibility with complex matrices. We believe the microfluidic fluidized bed-based approach presented here provides a new way to tackle high-performance immunoassays in fully automated protocols. Its versatility is based on the addition of new variables: the tunable porosity and arrangement of the magnetic beads. In particular, the extraction conditions can be tuned as a function of the diffusion constant of the analyte, while the enzymatic step can further be modified to improve the assay performances. This new technology is also compatible with a large range of biomarker concentrations as well as with sample volumes ranging from one to a few hundred µL.56. Moreover, we demonstrated its compatibility with challenging matrices of high protein content and no dilution while still ensuring a clinically-relevant sensitivity. We believe that this capability to enhance the performance of conventional assays while fully integrating complex sequential protocols make this approach a promising tool for future biomarker detection and quantification applications.We demonstrate here that it allows a fast detection of the cytokine IL-6 with a large dynamic range (10 pg/mL to 2 ng/mL), in less than 2 h, with an LOD in the picomolar range. The sensitivity achieved in this first proof of concept is applicable for instance to severe sepsis infection, where IL-6 levels can go up to a few hundreds of pg/mL in human serum samples. A LOD of 6 pg/mL for IL-6 spiked in buffer solution was achieved. This value is lower than the LOD achieved in experiments performed with the same reagents in on-bench conditions, and better overlaps with clinical rangesSupplementary Information.


Curcilio sayi) is an emergent pest of chestnuts in the United States that can cause multifaceted damage and has limited management options. We explored the damage caused by C. sayi in a commercial chestnut orchard. Additionally, we evaluated potential management options for biological control. We found that C. sayi emerged from infested chestnuts more than four weeks post harvest and, in some cases, single nuts can host more than 10 C. sayi larvae. We also found that nut weight continues to decline even after C. sayi larvae have emerged from the chestnuts. Specific strains of entomopathogenic nematodes increase the mortality of C. sayi larvae. Biological control using entomopathogenic fungi and entomopathogenic nematodes could be complementary approaches to managing this pest and reduce C. sayi populations and chance of damage.The lesser chestnut weevil , can cause irreparable damage to chestnuts through direct consumption and/or introduction of secondary pathogens. With the resurgence of blight resistant American Chestnut plantings both for commercial production and for habitat restoration, C. sayi has become a similarly resurgence pest. Here, we investigated the nature and extent of C. sayi larval damage on individual nuts and collected harvests with an eye toward the quantifying impacts. Next, we explored management options using biological control including entomopathogenic fungi and entomopathogenic nematodes. Nut damage from C. sayi can be extensive with individual nuts hosting several larvae, larvae emerging from nuts several weeks post harvest, and nut weight loss even after C. sayi have emerged from the nut. Applications of entomopathogenic fungi reduced chances of chestnut infestation, while certain strains of entomopathogenic nematodes increased the probability of C. sayi larval mortality. Understanding C. sayi damage and exploring biological control management options could be a useful tool in the effective management of this resurgent pest.The lesser chestnut weevil,  Castanea dentata) was once the foundation species for forests throughout Appalachia along the east coast of the United States. Occupying at least 50% of the basal area across 800,000 square kilometers, American Chestnut trees were once estimated to have numbered more than 4 billion in the early 1900s [American Chestnut  in the early 1900s, American Chestnut trees suffered a precipitous and rapid decline. By 1960, American Chestnut trees were almost completely absent from the canopies of eastern forests [Curculio sayi).With the introduction of chestnut blight  in upstate New York. This commercial stand was composed of a blend of mature (15+ years old) American Chestnut Hybrids planted approximately 7 m on center and forming a complete canopy after leafing out. Catkins (flowering) occurred in mid to late June and nut drop began in late September to early October. The soil type was Elnora Loamy Fine Sand.To evaluate the damage and management of C. sayi, chestnuts were collected within 24 h of nut drop and brought to the lab in two different cohorts.To evaluate the damage caused by For the first cohort, fifty chestnuts from each of four treatments  were placed individually into 50 mL Falcon Tubes. Each of these nuts were evaluated every 24 h and the number of larvae that had emerged from the nuts counted.For the second cohort, chestnuts sufficient to fill a 1 L plastic planting container were collected from the control treatment (described below). These planting containers were perforated on the bottom to allow for larval egress after emergence from the chestnuts. Ten replicates of these containers were collected. Every 24 h for 45 days these containers were evaluated. The number of larvae that had emerged were counted and weighed. The total weight of the nuts in the container were also weighed.C. sayi larvae, we evaluated the mortality of late instar C. sayi larvae exposed to thirteen different strains of entomopathogenic nematodes collected from the east coast of the United States. Late instar C. sayi larvae were collected as they emerged from harvested chestnuts and placed individually into wells of ELISA plates lined with 1 cm diameter filter paper ). Each of these wells received 200 µL of liquid; controls received water only while treatments received a nematode solution containing 1000 infective juveniles of the appropriate strain per mL for approximately 200 nematodes per larvae. Treatments were replicated 24 times and controls replicated 96 times across ELISA plates. C. sayi mortality was monitored for 10 days.To evaluate the ability of entomopathogenic nematode strains to control C. sayi, we evaluated both the application of entomopathogenic nematodes (EPN) to the soil and application of entomopathogenic fungi (EPF) to the trunks of chestnut trees. To do so, we utilized a two factor, full factorial design with four treatments: EPN Only, EPF Only, Both EPN and EPF, and the Control (neither EPN nor EPF). We utilized a randomized controlled block design and treated individual monitoring locations (approximately four trees) as replicates.To evaluate the viability of biological control techniques for the management of Steinernema feltiae and Heterorhabditis bacteriophora both collected from upstate NY and reared in Galleria mellonella larvae following previously established methods [Entomopathogenic nematodes were applied to the soil using a 15L backpack sprayer  at a rate of 120 million per acre. Two complementary species of entomopathogenic nematode were used:  methods . These sBeauveria bassiana, commercially available in Mycotrol ESO  was used as the entomopathogenic fungi at the recommended rate (1 quart/acre) on June 17th in both 2019 and 2020. The efficacy of this product was confirmed by sampling the solution immediately prior to the field application then subsequent application to G. mellonella larvae in the lab. Plots not receiving the EPF treatment instead received the same amount of the solution but without B. bassiana.Entomopathogenic fungi were applied to the trunks of each tree in the treatment in a 0.5 m band entirely circling the circumference of the tree, terminating approximately 1 m above ground level using a large paintbrush similar to whitewashing. C. sayi populations. Pyramid traps  are designed to attract newly emerging C. sayi adults as they climb adjacent vertical objects. These traps were staked to the ground approximately 0.5 m from adjacent tree trunks. Trunk Traps  were affixed to the tree trunks at breast height (1.35 m) such that the mesh screen of the trap directly abutted the trunk of the tree. These traps are designed to arrest and C. sayi adults ascending the trunks of the trees. These traps were emplaced in late April before the start of each season and monitored biweekly.The efficacy of these treatments was evaluated using pyramid and trunk traps to monitor adult C. sayi late instar larvae (that had emerged from collected chestnuts within 48 h) were placed into each microcosm and sealed inside with a mesh cap on the buried buckets. This arrangement ensured that the C. sayi could not escape the microcosms but that other microorganisms and water could move in and out via the soil.Microcosms consisted of 20, five gallon (18.93L) plastic buckets into which 15–20 large diameter (about 7cm) holes were drilled at the sides and bottom of the bucket and then covered with a fine mesh screen (1000 micron). These microcosms were placed adjacent to the commercial chestnut orchard by excavating 20 individual holes for each microcosm, placing the buckets in the holes, then backfilling the soil into each bucket. The soil was allowed to resettle for one week then 25 S. feltiae and H. bacteriophora in a 1:1 ratio at 1000 IJs/ml for each species . Entomopathogenic fungi only treatments received 500 mL of Mycotrol ESO B. bassiana solution . Treatments receiving both entomopathogenic nematodes and entomopathogenic fungi received the same amount of total volume with both EPN and EPF treatments mixed. The application of treatments occurred one week following introduction of the C. sayi larvae.Each of the four treatments described above were replicated five times in a randomized controlled block design. Each microcosm in each treatment received 500 mL of solution. Control microcosms received 500 mL of water. Entomopathogenic nematode-only treatments received a 500 mL of a solution containing C. sayi larvae and application of treatments accomplished by late November 2019. Microcosms remained unperturbed until November 2020 when they were removed, taken to the lab, and carefully excavated. As a first step in the excavation, the covering screen was removed and the surface examined for emerged C. sayi adults. Following this initial examination, the soil was carefully excavated and delicately passed through a fine mesh seive to extract any remaining C. sayi adults, pupae, or larvae.Microcosms were initially emplaced in late October 2019 with the introduction of C. sayi larvae was visualized using histograms and density distributions then modeled using generalized additive models.Nut damage by emerging C. sayi larvae resulting from nematode infection was evaluated using logistic regression considering the interaction between nematode strain and days post inoculation as factors. Best fit models were chosen after consideration of all potential interactions, residual analysis, goodness of fit metrics, and likelihood ratio tests. Post-hoc treatment comparisons were conducted using the Dunnett method of contrasting treatments with the control.Mortality of C. sayi management strategies was evaluated using poisson regression models for trap catch and emergence from microcosms. Logistic regression was used to evaluate nut infestation from respective treatments. Best fit models were chosen after consideration of all potential interactions, residual analysis, goodness of fit metrics, and likelihood ratio tests. Post-hoc treatment comparisons were conducted using custom contrasts for desired comparisons and the Bonferroni correction for adjusting the family-wise error rate.The efficacy of All analysis was conducted in R version 4.2.1  using RSC. sayi emerged from individual chestnuts at varying rates and over an extended period of time. Out of the chestnuts with emergent larvae, eight had only one larva emerging in the time under observation (C. sayi larvae that emerged under the period of observation. More than three quarters of chestnuts (75.8%) with larval emergence had more than one larva emerging over the observation period.Larval ervation A. A few C. sayi began emerging within a few days of the field collection of nuts dropped in the previous 24 h  significantly explained adult C. sayi trap catch . Chestnuts from the treatment receiving only entomopathogenic fungi had a significantly lower chance of weevil infestation both as a main effect and compared to the control treatment . Treatments receiving entomopathogenic nematodes only had a significantly lower (p = 0.007) number of weevils recovered compared to the EPN + EPF treatment  tended to occur between 10 and 24 days post harvest, the emergence of the first weevil from an individual chestnut occurred as early as a few days following harvest and as late as 42 days post harvest. This long time period for larval emergence was corroborated in observations of planting containers of chestnuts with the last larva emerging 31 days post harvest.Emergence of C. sayi emergence suggests that post harvest control strategies may need to be adapted. A practice that involves waiting for two weeks (14 days) to determine which nuts are not infested does not seem adequate. Even nuts with no emergence after 14 days may still contain active larvae that could emerge potentially even three weeks later.The distributed nature of C. sayi in buckets of chestnuts occurred at six days post harvest and dropped to low levels by day 16 when the cumulative weight of the larvae flatlined. Despite very little emergence occurring after day 16, nut weight continued its almost linear decline.Waiting six weeks (42 days) to evaluate larval emergence and identify healthy nuts is also not an ideal option. Irrespective of time to market considerations, nut weight appears to decline linearly with time. This is true even after most larvae have left the nuts. Peak emergence of C. sayi consumption of nut meat. A potential culprit is a bacterial/fungal complex associated with C. sayi larval tunnels inside the nuts. Feeding by C. sayi larvae open channels in the chestnut, which are colonized by potentially harmful microorganisms such as Aspergillus fungi [C. sayi larvae have emerged from the nut. Their presence could be a contributing factor to the continued decline of nut weight over time. Independent of the causes, the more time between harvest and market delivery of chestnuts, the less chestnut material is available.This decline suggests that other factors are at play in decreasing nut weight, not solely us fungi ,9,29,30.C. sayi, management techniques that address burgeoning populations are critical. Applications of entomopathogenic fungi to trunks of mature chestnut trees significantly reduced the adult C. sayi trap catch compared to the control.Because of the losses caused by C. sayi moving to the tree canopy. Second, applications of entomopathogenic fungi could have had an effect of C. sayi mortality. While not immediately effective in this regard, insects coming into contact with entomopathogenic fungi can be colonized by this fungus and die in a matter of days from a systemic fungal infection.This reduction in trap catch could potentially be attributed to two possible factors. First, the applications of entomopathogenic fungi to the trunks of chestnut trees could have potentially served as a repellent to adult C. sayi trap catch was also reflected in the chance of weevil infestation in collected chestnuts. Chestnuts collected from the EPF treatment had a lower chance of being infested with C. sayi larvae than other treatments. This suggests that the effect of application of entomopathogenic fungi, translates into a reduction in weevil infestation of harvested chestnuts.Irrespective of the mechanism, the reduction in adult C. sayi trap catch and chance of infestation, in each case, treatments of entomopathogenic fungi lowered the respective response compared to the entomopathogenic nematode-only treatments.Treatments receiving both entomopathogenic nematodes and entomopathogenic fungi did not respond the same as entomopathogenic fungi only treatments. While these treatments were comparably lower in terms of adult C. sayi larvae. Although the strains chosen in this study were selected more for their ability to survive northern winters as opposed to virulence alone, they still increased the probability of larval mortality in laboratory trials. In particular, in one of the species we used, (S. feltiae) can significantly increase the probability of mortality compared with controls. While it is entirely possible that applications of entomopathogenic nematodes had no effect on C. sayi populations, it is also possible that the effect might be lagged. The drastic drop in C. sayi populations between years might be the result of the efficacy of entomopathogenic nematodes, which are are highly effective mobile agents that seek out weevil larvae. A similar effect has been observed in applications of entomopathogenic nematodes to control the pecan weevil (Curculio caryae); repeated applications of entomopathogenic nematodes resulted in the improved control of C. caryae in year two [The lack of response of the entomopathogenic nematode treatments in the field was surprising given that many entomopathogenic nematode strains significantly increased the probability of year two .C. sayi as well, seem to demonstrate a staggered approach to cohort development with potentially multi-year periods in the soil as eclosed adults, timing in using entomopathogenic nematodes could be critical [C. sayi in the soil are in nematode-resistant pupal cells, as more nematode resistant adults [Because chestnut weevils in general, and likely critical ,32,33. Et adults ,34.C. sayi are emerging from chestnuts and burrowing into the soil as larvae prior to establishing protections from soil organisms like entomopathogenic nematodes. The efficacy of these applications would not appear, however, until the next season, when the impact of this mortality on the emerging cohort would become apparent. If entomopathogenic nematodes were effective, we would expect this to become apparent over the course of two to three years as we began to see in this study and as has been observed in other field settings [C. sayi when introduced to the soil as larvae.In contrast, applications of entomopathogenic nematodes at the end of the season might be more effective. At the end of the season, settings ,36. In aC. sayi infestations in commercial chestnut orchards. This dual approach has been evaluated in other similar systems with the greater chestnut weevil (C. elephas) [C. sayi populations over the longer term. There might even be a synergistic effect to this approach that emerges over the course of three to five years. This is the focus of future work.Together, entomopathogenic nematodes and entomopathogenic fungi could present an option for the biological control of elephas) . Applica


Our study is a randomized trial in policing confirming that intensive training in procedural justice (PJ) can lead to more procedurally just behavior and less disrespectful treatment of people at high-crime places. The fact that the PJ intervention reduced arrests by police officers, positively influenced residents’ perceptions of police harassment and violence, and also reduced crime provides important guidance for police reform in a period of strong criticism of policing. This randomized trial points to the potential for PJ training not simply to encourage fair and respectful policing but also to improve evaluations of the police and crime prevention effectiveness. Can police be trained to treat people in fair and respectful ways, and if so, will this influence evaluations of the police and crime? To answer these questions, we randomly allocated 120 crime hot spots to a procedural justice (PJ) and standard condition (SC) in three cities. Twenty-eight officers were randomly assigned to the conditions. The PJ condition officers received an intensive 5-d training course in the components of PJ . We used police self-report surveys to assess whether the training influenced attitudes, systematic social observations to examine impacts on police behavior in the field, and arrests to assess law enforcement actions. We conducted pre and post household surveys to assess resident attitudes toward the police. Impacts on crime were measured using crime incident and citizen-initiated crime call data. The training led to increased knowledge about PJ and more procedurally just behavior in the field as compared with the SC condition. At the same time, PJ officers made many fewer arrests than SC officers. Residents of the PJ hot spots were significantly less likely to perceive police as harassing or using unnecessary force, though we did not find significant differences between the PJ and SC hot spots in perceptions of PJ and police legitimacy. We found a significant relative 14% decline in crime incidents in the PJ hot spots during the experiment. There has been a growing call for reform of the police , 2, ofte4Procedural justice (PJ) involves fair and respectful treatment of people by police  . Scholar12SI Appendix, section S1). We first carried out the experiment in Tucson (1 July 2017 to 31 March 2018) as a test of whether the training and treatment could be delivered as intended. We then moved to Cambridge (2 February 2019 to 31 October 2019) and Houston (12 October 2019 to 10 July 2020). We compare two conditions, PJ hot spots patrolled by officers that received training  and were encouraged to build trust and reduce crime in hot spots and standard-condition (SC) hot spots where officers focused just on reducing crime.In this paper, we present findings from a three-city  randomized field trial that tested whether PJ training would impact police officer behavior, hot-spot residents’ perceptions of police, and crime. The intervention period was 9 mo in each city. We also began the study in a fourth city, but COVID-19 led to implementation of treatment for only half of the study period and prevented key data collection . We required that the hot spots had at least 15 residential dwelling units to ensure a sufficient sampling frame for our community survey. We selected 40 crime hot spots in each city at least one street segment distant from each other. A power analysis shows a minimal detectable standardized effect size of 0.23 for nondirectional tests and 0.20 for directional tests, suggesting that the study was sufficiently powered to identify small to moderate program impacts for outcomes at the hot-spot level .The focus of the intervention was on crime hot spots, which were defined as high-crime residential street segments, intersection to intersection. We used crime incident data, which represent crimes reported by the police, and citizen-initiated crime calls to identify hot spots . In the selection year, the sample hot spots in Tucson had a mean of 133.98 crime incidents  and 216.02 citizen-initiated crime calls ; Cambridge hot spots had a mean of 17.38 crime incidents  and 68.73 citizen-initiated crime calls ; and Houston hot spots had a mean of 27.78 crime incidents , and 85.8 citizen-initiated crime calls . The demographic characteristics of the hot spots based on the community survey (see Community Survey) are reported in SI Appendix, Table S7. The respondents in our community survey of the crime hot spots were 40% Latino in Tucson, a majority white in Cambridge, and a majority Black in Houston.After selecting the crime hot spots, they were randomly assigned, within statistical blocks defined by crime levels, equally to the PJ and SC groups .In Tucson and Houston, eight officers participated in the program, and in Cambridge 12 officers participated. All officers worked in a patrol role prior to the project start. Once we received a list of the officers and their characteristics, we matched officers in pairs in terms of background, including race and ethnicity, gender, and experience. We then randomly allocated one officer in each pair to the PJ group and one to the SC group. There were no large or statistically significant differences in background characteristics or views of PJ between the groups at baseline . The first day of training included an overview of hot-spots policing, police legitimacy, and PJ. Day two focused on each of the four elements of PJ , as well as the importance of historical context in understanding trust in police. Day three included discussions of scenarios as well as a role-playing exercise where officers had a chance to practice using PJ in interactions. Day four applied the ideas of PJ to working with diverse populations and those with behavioral health problems. The second half of day four introduced the project hot spots and covered applying PJ to hot-spots policing tactics. Finally, day five incorporated plans for officer supervision and officer forms . The training protocol is detailed in SI Appendix, section S1.3. We also followed up with a 1-h, in-person refresher training for PJ officers in each city about 3 mo after the initial training. An officer survey conducted before and after training suggests that officer knowledge of components of PJ significantly improved after the training . The SC group received a half-day training on hot-spots policing tactics and project data collection, which was also part of the training of the experimental condition.Guided by existing PJ training models, we developed an intensive training program for the PJ officers that was delivered over 40 h . In Tucson the PJ hot spots received about 123,911 min of individual police presence, in Cambridge about 136,673 min, and in Houston (where there were often long distances between the hot spots) about 62,974 min. Looking at the SC sites, we gained similar estimates . While the project officers responded to citizen-initiated calls for service at their assigned hot spots, they spent a substantial amount of time focusing on proactive police activities.Treatment dosage was measured using activity logs describing the amount of time the study police officers spent at the project sites . While these estimates rely on available data, they suggest that a substantial part of policing in the PJ group hot spots was provided by the project officers. Outcomes are similar for the SC group officers .Because the hot spots continued to receive patrol response to emergency calls when project officers were not present, we sought to identify the proportion of overall policing at the PJ hot spots that was provided by PJ officers. To estimate this, we used police management systems, which track police responses to calls for service and officer-initiated activities. The Cambridge data identified police officer minutes directly by officer. Houston provided similar information, but only one officer was listed for each event. Tucson did not include the number of minutes officers spent on site—this was calculated based on data provided by the police department for project officers in the 6 mo preintervention. We estimate that 50.56% of total police time at the hot spots came from experimental officers in Cambridge, 61.23% in Houston, and 48.50% in Tucson , which provide an easily interpreted metric for understanding the magnitude of study findings. Cohen provided standard metrics for small (d = 0.20), moderate (d = 0.50), and large effect (d = 0.80) sizes, though reviews in criminology suggest that these metrics may be overly conservative  to assess whether the police officers acted in procedurally just ways, arrests as a measure of law enforcement activity, community survey data to assess perceptions of the police, and crime incidents and citizen-initiated crime calls to examine impacts on crime. This project was approved by institutional review boards at Arizona State University and Northeastern University. In order to provide a comparative review of study outcomes, we computed Cohen’s ervative , 22.SI Appendix, Fig. S3) were assigned by the research team to ride with one officer for an entire shift, varying which officers rode with which observers and the days of the week each officer was observed . We conducted 129 ride-alongs in the three cities (66 in the PJ group and 63 in the SC group). PJ and SC ride-alongs were balanced in the days of the week they were conducted, the officers who were observed, and the number of shifts per officer observed . The ride-alongs yielded 334 encounters with 508 interactions between police officers and citizens. The encounters did not always occur at the hot spots—for example, officers sometimes followed cars outside the hot spots or made an on-site stop of suspicious persons or vehicles riding to or from the hot spots . While we think that our assessment of PJ should be based on all officer contacts, we also analyzed data only for the hot spots as a sensitivity analysis.Trained observers , City is a dummy variable for city, and citizen interactions with police are nested within encounters (ju) nested within officers (kr):In analyzing these data, we used a mixed-effects regression modeling approach in which city and treatment group are fixed effects and police officer and encounter are random effects . The model may be expressed as follows where SI Appendix, Table S17), we produced an overall PJ score and indices for the four main PJ components: voice, neutrality, dignity and respect, and trustworthy motives. Imputed values were generated for voice missing scores when creating the overall PJ measure because in some encounters citizens chose not to respond to officers, so observers were unable to assess whether the officer was an active listener during these encounters . Because disrespectful encounters have been found to be particularly influential on perceptions of police . We analyzed these data at the police officer level including treatment group, city, and log of preintervention arrests as independent variables. We model arrest outcomes using the standard log-link function for a negative binomial regression (because of overdispersion in the intervention period) where y represents arrests during the intervention period, Group refers to officer group (PJ group =1), City is a dummy variable for city, and PreArrests is an indicator of the number of arrests an officer made in the 6 mo prior to the start of the intervention:We collected data on arrests made by project officers in the 6 mo prior to the intervention and during the intervention period , from which dwelling units were randomly sampled with the goal of gaining seven surveys per hot spot . Door-to-door residential surveys were conducted before and after the intervention period. Trained field researchers, who were not told whether they were in a PJ or SC hot spot, interviewed the first adult resident contacted at a household who had lived on the street for at least 3 mo. The surveys were conducted face-to-face with field researchers reading the survey to participants after obtaining informed consent. Respondents were compensated $20 for their participation. The response rate (number of surveys completed/number of households sampled) was 33.53% for the preintervention survey across the three cities. An average of seven surveys were completed on each street in the preintervention survey. Additional details regarding the survey methodology are provided in SI Appendix, section S1.8 and Fig. S6.The first step in the community survey was to develop a census of households on each street . We used contact information provided in the preintervention survey, as well as mailing postcards to sampled households, and purchasing phone numbers from third-party providers . These methods yielded a much lower response rate than the preintervention survey (17.01%). We did not find significant differences in background characteristics between the pre- and postintervention surveys in Houston for gender, race, age, having a college degree, or household size, though postintervention respondents were significantly more likely to own their homes (59.6% versus 46.2%)—reflecting the increased likelihood of identifying phone numbers for homeowners . While we were able to collect between 229 and 329 surveys pre and post for Tucson and Cambridge and 277 surveys preintervention in Houston, we were only able to collect 109 surveys postintervention in Houston.Data collection in the postintervention survey in Tucson and Cambridge followed protocols similar to the preintervention survey, with the difference that we began sampling with preintervention respondents. For the postintervention survey, the response rate in Tucson and Cambridge was still high at 35.84%. Because of COVID-19, we were not able to carry out door-to-door surveying postintervention in Houston and relied on telephone surveying . We also carried out the analyses involving single response measures using multilevel mixed-effects ordered logistic regression models .As a sensitivity test, we also analyzed data at the respondent level and estimated multilevel mixed-effects linear models with probability weights . Police legitimacy was measured both in terms of attitudes toward police on the street where the respondent lived , as well as for the city overall . All questions used four-item Likert scales (strongly disagree to strongly agree) where higher values indicate more positive views about police.The process model of PJ proposed by scholars assumes that when the police behave in more procedurally just ways people will respond by assessing police authority as more legitimate , 10, 30.We also asked residents about their perceptions of police in regard to two issues that have received prominence in recent criticisms of policing. The first asked whether the respondent believed that the police harass or mistreat people on their street. The second asked whether the respondent believed the police on their block use more force than they have to. These items are also measured using a four-item Likert scale.We employed one-tailed tests of statistical significance for these analyses as the treatment has a strong presumption of increasing PJ and legitimacy and reducing perceptions of harassment and use of police violence.SI Appendix, section S1.10). Our measure for total crime incidents and total citizen-initiated crime calls included violent, property, drug, disorder, domestic, and other crimes . We did not examine specific crime categories because the Ns for analysis at the hot spot level become relatively small. We modeled crime outcomes using the standard log-link function for the negative binomial distribution because of overdispersion in crime counts in the intervention period. In the regression models estimated, y indicates the count of crime incidents or calls for service either during or after the intervention (depending on the model), Group is the treatment group the hot spot was assigned to, City is a dummy variable for city, Block is a dummy variable for statistical block used for hot spot randomization, and Pretest indicates the number of incidents or calls on the block in the six months preintervention:Crime incident data are ordinarily used by police for describing crime in communities but may underreport crime since police officers must write up a report that a crime has occurred. Citizen-initiated crime call data may overstate the extent of crime problems, since people may not always have accurate information about events they observe. Additionally, in programs that emphasize community engagement, there is some evidence that citizen reporting may be inflated relative to standard policing conditions . We asseWe compared the intervention period to a 6-mo preintervention period and the preintervention period to a 6-mo postintervention period. We employed two-tailed significance tests because there is not a strong presumption of the direction of treatment influence. While PJ advocates argue that procedurally just policing could reduce crime by enhancing legitimacy and increasing compliance with the law , 32, othP < 0.01), show neutrality (P < 0.05), and demonstrate respectful behavior (P < 0.05) in observed interactions. Cohen’s d values range between 0.22 and 0.39, suggesting standardized small to moderate treatment impacts . Although our index of trustworthy motives fails to achieve statistical significance in these analyses, the impact is in the expected direction. The overall PJ index is strongly significant (P = 0.001), with the PJ group showing more procedurally just behavior (Cohen’s d = 0.39). Interactions involving SC group officers were significantly more likely to include disrespectful behavior (P = 0.01). In this case, Cohen’s d is moderate with a value of −0.51. Looking at PJ measures evidencing significant outcomes, we do not observe statistically significant variation across the three cities .PJ group officers were significantly more likely to give people voice . We also estimated the overall PJ index including only nonimputed values for voice . These analyses produced very similar findings to those reported in We report in P < 0.001) to make arrests during the experiment than the SC group officers . The IRR suggests a relative reduction in arrests of more than 60%. Arrest behavior does not vary significantly across the three cities , though few arrests were made in Cambridge .We also find that the PJ group officers were much less likely  and using more force than necessary (P < 0.05). The effect sizes are small to moderate with a Cohen’s d value of −0.47 for harassment and −0.34 for police violence. We did not observe significant variability across the three cities for harassment or police violence . Analyses conducted using multilevel mixed-effects linear regression models , or multilevel mixed-effects ordered logistic regression models  and partial proportional odds models  produced very similar results.When comparing post- to preintervention surveys, the PJ and SC conditions differed little in regard to community perceptions of PJ or police legitimacy . Given the increased number of arrests in the SC group (which would be associated with crime incidents), one explanation for this finding might be that it is due to increased enforcement behavior. However, in Cambridge, where few arrests were made overall, the observed treatment effect on crime incidents is larger than the average observed across the cities . There was a 10% relative reduction in crime incidents observed in the PJ group hot spots comparing the pre- to postintervention period . Analyzing the citizen-initiated call data, we do not observe significant outcomes. However, the observed effects are in the direction of relative crime reductions in the PJ hot spots .We report in riod see , but theSI Appendix, section S2.7; officer surveys and SSOs were collected before pandemic restrictions). Following our results described above that there were not significant treatment-by-city interactions for these analyses, the results excluding Houston showed the same general pattern as the full three city models .Given that the COVID-19 pandemic was ongoing in Houston for part of the intervention period and the postintervention period, we also estimated our models excluding the Houston data for arrests, the community survey, and crime (Can we improve police behavior at crime hot spots, where proactive policing interventions are carried out? The answer from our study is that we can through intensive police training in PJ. At the same time, such training is found to lead to fewer arrests, and to people reporting that the police are less violent and less harassing of people who live on their street. The fact that we also observe crime prevention benefits, at least in comparing the preintervention period with the intervention period is noteworthy, though that benefit is modest and only in regard to crime incident data.The 2018 review of the National Academies of Sciences Committee on Proactive Policing  found th9We also think that our study provides important insights regarding police training and its potential influence on police officer behavior. The National Research Council concluded in 2004 that “for many decades it has been assumed that more and better police training leads to improved police performance,” but that “few studies evaluate the impact of training programs on actual performance on the job” . More thWhile we think our study strongly advances our knowledge about PJ policing, it is important to recognize that our findings are drawn from a trial in three specific cities, and in one city COVID-19 restrictions were in place for part of the treatment period and the collection of survey data. While we were able to successfully implement the study in three cities, given the diversity of America’s urban areas, replications of our experiment in other contexts are needed. We note as well the importance of studies in which all police service is provided by specially trained officers. One possible explanation, for example, for the lack of influence of treatment on perceptions of police legitimacy may be the continued presence on these streets of untrained patrol officers responding to emergency calls to the police. With these limitations in mind, our randomized trial points to the potential for PJ training not simply to encourage fair and respectful policing, but also to improve evaluations of the police and crime prevention effectiveness.Supplementary File


Alphatorquevirus), a genomovirus (genus Gemykolovirus), and an unclassified papillomavirus were identified in four human nasopharyngeal swabs, and one was positive for influenza A and one for influenza B virus. The influenza B virus-positive sample had a coinfection with an anellovirus and a papillomavirus.The genome sequences of three anelloviruses (genus  To identify associated DNA viruses in these samples, 200 μL of the buffer from the lateral flow assay was used to extract viral DNA with the high pure viral nucleic acid kit . Circular DNA was amplified by rolling circle amplification (RCA) using an Illustra TempliPhi kit . The RCA products were used to prepare libraries using a TruSeq Nano DNA kit . The 2× 150-bp libraries were sequenced on a NovaSeq6000 sequencer at Psomagen Inc. (USA). The reads were trimmed using Trimmomatic v0.39 (e BLASTn . In sampe BLASTn .Anelloviridae family (MW679005), and torque teno virus AZ7_4 (MW679006) identified in the nasal pharyngeal swabs all belong to the genus Alphatorquevirus and species Torque teno virus 24, Torque teno virus 29, and Torque teno virus 19, respectively (Anelloviruses are circular single-stranded DNA (ssDNA) viruses (2.0 to 3.9 kb) . They are family , torque ectively .Fusarium graminearum gemytripvirus 1 which is a tripartite virus  shares ~89.6% genome-wide nucleotide identity with tortoise genomovirus 13 (MK570213) (Gemykolovirus and species Gemykolovirus gopha2 based on the classification guideline for Genomoviridae (–Genomoviruses are circular ssDNA viruses (2 to 2.4 kb) that encode bidirectionally transcribed ORFs, capsid protein (CP), and a replication associated protein (Rep), with the exception of te virus –10. Althrotiorum  and Fusaminearum . GenomovK570213)  (13) andoviridae –18, and Firstpapillomavirinae and Secondpapillomavirinae)  identified in the swab shares ~97% nucleotide identity with HPV-mSK_175 (MH777317) from a human skin swab (KC113191) from an oral swab (Papillomaviruses are circular double-stranded DNA viruses (~5.7 to 8.6 kb) and are classified within two subfamilies (virinae) . Membersen genes . Human pkin swab  Fig. 1)Firstpapiral swab ; neitherSample collection was part of routine clinical care which is approved by Arizona State University Institutional Review under study identifier (ID) number STUDY00008985.MW679002, MW679003, MW679004, MW679005, and MW679006. Raw reads have been deposited in SRA project number PRJNA701833 with SRA accessions SRR13720055, SRR13720056, SRR13720057, and SRR13720061.The sequences of the viruses identified in this study have been deposited in GenBank with accession numbers


Citrus medica L. var. sarcodactylis) contains different bioactive compounds, and their effects remain unclear. Therefore, the structural and bio-function of bergamot dietary fiber were investigated. A sequential extraction procedure was utilized to obtain soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) from bergamot. The main monosaccharide in SDF and IDF is arabinose. SDF had a porous structure, which enhanced the water and oil holding capacity, as well as the cholesterol and glucose adsorption capacity, which was superior to that of IDF. In db/db diabetic mice, SDF and IDF regulated glucose tolerance and controlled blood glucose levels. Reduction of serum total cholesterol, triglycerides, and low-density lipoprotein cholesterol in SDF and IDF could be observed. In summary, SDF and IDF from bergamot effectively promoted health in patients with diabetes.Bergamot ( Citrus medica L. var. sarcodactylis) belongs to the citrus family , a carbohydrate-based polymer, is mostly derived from edible plants such as vegetables and fruits . DF is cType 2 diabetes mellitus (T2DM) is characterized by insufficient insulin production or the inability to absorb it for glycemic regulation in the human body . In addiDF may aid in reducing appetite, dietary intake, obesity risk, blood sugar levels, and cholesterol levels . Gastricin vivo. This study offers helpful information on the bioactive properties of bergamot and related DFs.In this study, SDF and IDF were extracted from bergamot and their physicochemical properties, water- and oil-holding capacity, cholesterol and glucose adsorption, and glycemic regulation were characterized Bergamots were obtained from Huizhou . In addition, papain, heat-stable α-amylase, cellulase, neutral protease, and saccharifying enzyme were purchased from Guangzhou Dongju Co., Ltd. . Other chemicals and reagents used were commercially available.w/v) was added to the mixture, which was then treated for 30 min at 60°C. Four liters of 95% (v/v) ethanol were added when the mixture was cooled to 25°C to precipitate the SDF for 12 h. The method for the extraction of IDF was also used in a previous study (w:v), centrifuged at 1,500 × g for 15 min, and the sample was precipitated. At 70°C, pH 5.5, and 0.6% enzyme dosage, α-amylase was applied to the precipitate for 80 min. To obtain the IDF, the residue was finally filtered and dried.Bergamots were cleaned, sliced, and dried at 55°C for 24 h. Then, the dried bergamots were ground into flour in a crusher and kept at 4°C. SDF extraction was conducted as previously described . Brieflyus study . BergamoThe composition, such as ash (AOAC 920.138) and protein (AOAC 992.23), was determined as previously described .The SDF and IDF were sprayed with metal, and their spatial structures were analyzed by SEM  at a magnification of × 200.−1 frequency range, the bergamot SDF and IDF were ground with potassium bromide powder (KBr).An FT-IR spectrophotometer  was used to measure the FT-IR spectra of bergamot SDF and IDF. Before being formed into pellets (5 mg DF sample/200 mg KBr) for FT-IR scanning in the 4,000–400 cmThe assay for monosaccharide components was conducted as previously described by Xiong et al. with some modifications . StandarThe WHC of the bergamot SDF and IDF was measured as described previously  with minThe OHC of the bergamot SDF and IDF was determined as described previously  with minThe CAC of the bergamot SDF and IDF was determined using the o-phthalaldehyde method  with minThe glucose adsorption capacity (GAC) of the samples was evaluated as previously described .n = 6 in each group) for analysis: (1) non-diabetic lean littermates ; (2) db/db mice treated with vehicle (db group); (3) db/db mice treated with metformin 200 mg/kg BW/day (met group); (4) db/db mice treated with SDF 800 mg/kg BW/day (SDF group); and (5) db/db mice treated with IDF 800 mg/kg BW/day (IDF group). Throughout the experiments, the mice were fed a standard chow diet and tap water ad libitum. The duration of oral administration was 8 weeks. All experimental protocols were performed in accordance with the regulations of the Laboratory Animal Care and Use Committee (No. HZYX20220222641901).This method was conducted as previously described . BrieflyLiver and pancreatic tissue samples were weighed or immediately snap-frozen in liquid nitrogen until use. The BW of each mouse was recorded every 2 weeks. The liver index was calculated according to a previous study . The micBlood glucose was determined using the OGTT. Specifically, after 12 h without meals, blood samples were taken from the tail vein and the levels of glucose were evaluated. Then, glucose was given orally to the animals at a dose of 2 g/kg BW, and blood was drawn from the tail vein every 30 minutes to measure the glucose levels. The area under the curve (AUC) was calculated using the measurement results .Total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) levels were determined using commercial reagent kits . All procedures were carried out by following the manufacturer's instructions.Different groups of mice were used to obtain liver and pancreatic tissue samples. The tissues were dehydrated, flushed with PBS, fixed with 10% formalin, embedded in paraffin, and sectioned. Finally, the sections were stained with hematoxylin and eosin. All images were obtained using a TE 2000 fluorescence microscope  .t-tests for two group, and by Tukey's multiple comparisons test with one-way ANOVA for multiple group comparisons; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; NS, not significant.Data are expressed as means ± standard deviation (SD) of three replicates. All graphical representations were generated using GraphPad Prism software . The data were analyzed by Student's Images of the SDF and IDF ( × 200) were used to show the morphology and size of the SDF  and IDF In contrast, IDF was flaky and schistose, with a compact texture. However, this porous structural feature of DF is conducive to immobilizing the molecules in the network structure or holes, enhancing the binding ability of DF .−1, attributed to the O-H stretching vibration of hemicellulose  of bergamot SDF and IDF were 38.12 ± 2.94% and 45.38 ± 3.52%, respectively. The SDF and IDF chemical compositions were analyzed . The proThe monosaccharide compositions of SDF and IDF are shown in Previous research has demonstrated that an arabinose- and rhamnose-containing polysaccharides may lower blood glucose levels in diabetic mice, and that arabinose supplementation can lessen glucose peaks and postpone insulin peaks . Howeverin vitro and could be utilized by the gut microbiota in the intestinal micro-environment to prevent diabetes.Due of its uneven, loose, and porous surface, DF has a high WHC . After ap < 0.05, The average charge density, hydrophobicity, physical properties, and preparation procedures all influence the OHC of DF . The OHCThe CAC of DF is classified as either physical or chemical adsorption. Physical adsorption is primarily affected by DF particle size, porosity, and temperature, whereas chemical adsorption is influenced by DF charge and hydrophobic groups . The CACp < 0.05). Because glucose may attach to the DF network structure and limit interaction with the human digestive tract to produce a physical barrier and delay glucose diffusion, the GAC of DF is connected to its physical features  and IDF groups (p < 0.01) compared with the db group . Specifically, blood glucose levels in the SDF and IDF groups fell to 24.93 ± 1.99 and 22.63 ± 2.27 mmol/mL, respectively, and were lower than the values in the db group (32.77 ± 0.90 mmol/mL). The db/db mice exhibit severe symptoms of hyperglycemia . Conversely, unlike in db/db diabetic mice, the AUC values of SDF and IDF groups from the OGTT in db/db diabetic mice decreased significantly (p < 0.01).As shown in p < 0.05). In the SDF group, the insulin level was 43.29 ± 0.56 mIU/L, lower than that of the IDF group (44.37 ± 1.91 mIU/L); however, the difference was not significant (Insulin resistance is a defining feature of T2DM. The homeostatic index of insulin resistance (HOMA-IR) and insulin levels are shown in nificant .p < 0.0001) values, as opposed to the db group, demonstrating that metformin has a positive impact in decreasing insulin resistance. In contrast, HOMA-IR in the SDF and IDF groups was significantly decreased compared to that in the db group (p < 0.001), with no significant difference . Furthermore, compared to levels in the db group, TC was dramatically lowered (p < 0.001) in mice subjected to SDF and IDF treatment . LDL-C levels significantly decreased in mice treated with SDF (p < 0.01) and IDF compared to those in the db group (p < 0.01) (The LDL-C level was significantly higher in the db group than in the metformin group ( < 0.01) . These fThe body obtains metabolic energy by producing glucose via hepatic glycogen disintegration and converting free fatty acids generated by adipose tissue deterioration . When thp < 0.01) than that of the metformin group (p < 0.01) and IDF (p < 0.001) intervention. Excessive blood glucose may be the cause of fat accumulation in the liver and may be linked to the formation of dyslipidemia (However, the liver index of mice in db/db group was significantly higher (in group , whereasipidemia . Furtheripidemia . As showDiabetes and its complications are becoming increasingly common worldwide, seriously influencing the lives of human beings. It is a prevalent chronic metabolic condition defined by dysglycemia and dyslipidemia, both of which inevitably result in health issues such as eye, kidney, heart, and hepatic dysfunction. This means that maintaining blood glucose and lipid levels is vital for diabetes treatment and prevention.in vitro. Notably, these capabilities of SDF were comparatively more effective owing to its loose and polar structure to bind with oil, cholesterol, and glucose. SDF and IDF mitigate the symptoms of hyperglycemia and protect pancreatic tissue. Specifically, SDF can enhance the glycemic regulation ability of db/db diabetic mice and may enhance insulin sensitivity in the target organs to regulate glucose homeostasis. In vivo experiments also indicated that SDF and IDF effectively controlled blood lipid levels and protected liver tissue in db/db diabetic mice. Also, this study discovered that SDF can more effectively inhibit cholesterol micelle production in vitro and control blood lipid levels in vivo by lowering serum TC, TG, and LDL-C levels, which is important for cardiovascular health and body shape, particularly when consuming fried or fatty meals.In this study, the SDF and IDF were extracted from bergamot. Their chemical composition, structural characterization, physicochemical properties, and their effects on blood glucose control and lipid levels in db/db mice were investigated. The DF network structures are completely different. SDF is loose and has honeycomb-like holes and cracks, while IDF is flaky and schistose with a smooth surface and dense texture. In terms of monosaccharide composition, arabinose was abundant in monosaccharides in both SDF and IDF. SDF and IDF from bergamot exhibited different structures, which varied in their WHC, OHC, CAC, and GAC Although dysfunction of db/db diabetes mice is complicated in its physiology, our future work will explore its pathway and elucidate the regulatory mechanism of bergamot SDF and IDF. Overall, the outcomes of this study show that bergamot SDF and IDF might be used to create and test multivalent functional foods that could supplement existing dietary interventions.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by Hangzhou Laboratory Animal Operation Center of Yanxuan Biotechnology Company. Written informed consent was obtained from the owners for the participation of their animals in this study.JLi and HL: conceptualization and methodology. JLi: data curation and writing—original draft preparation. GL, JLa, and RZ: visualization and investigation. GX: supervision. CL: software and validation. QW and JLi: writing—reviewing and editing. All authors contributed to the article and approved the submitted version.This work was supported by Research and Application on Germplasm Innovation and New Variety Cultivation Technology of Subtropical Fruits with Characteristics in Western Guangdong and Key areas of Guangdong Province Research and Development Plan project in 2021.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.