Sensing of clinically relevant biomolecules such as neurotransmitters at low concentrations can enable an early detection and treatment of a range of diseases. Several nanostructures are being explored by researchers to detect biomolecules at sensitivities beyond the picomolar range. It is recognized, however, that nanostructuring of surfaces alone is not sufficient to enhance sensor sensitivities down to the femtomolar level. In this paper, we break this barrier/limit by introducing a sensing platform that uses a multi-length-scale electrode architecture consisting of 3D printed silver micropillars decorated with graphene nanoflakes and use it to demonstrate the detection of dopamine at a limit-of-detection of 500 attomoles. The graphene provides a high surface area at nanoscale, while micropillar array accelerates the interaction of diffusing analyte molecules with the electrode at low concentrations. The hierarchical electrode architecture introduced in this work opens the possibility of detecting biomolecules at ultralow concentrations. Here, the authors introduce a biosensing platform with multi-length-scale electrode architecture consisting of 3D printed silver micropillars decorated with graphene flakes. They demonstrate that this breaks a barrier to the biomolecule limit-of-detection, enabling detection down to femtomolar level. This limitation has created economic burdens that were apparent in worldwide ravage caused by the pandemic associated with the novel coronavirus2. Most importantly, the human cost of this limitation needs to be addressed with utmost urgency.One of the key requirements for the effective management and treatment of diseases is their detection at an early stage. This requires a timely detection of the relevant biomarkers at very low concentrations. Despite considerable progress in this area, sensing techniques of broad applicability that can detect a range of biomarkers at ultralow  concentrations with high throughput  are yet to be realized3. An obvious method to increase its sensitivity is the nanostructuring of the surfaces, which is expected to increase the surface area of the reaction8. Several nanostructured surfaces have been explored for this purpose such as DNA nanostructures9, Bi2S3 nanowire microfibers10, Au-Pd/MoS2-MWCNT nanocomposite11, NiCo2S4 nanoparticles12, and host-surface nanotexturing13. These approaches have led to a significant advance in the detection sensitivity. However, as pointed out in the seminal works by Sheehan and Whitman1 and others16, the nanostructures are typically grown on two-dimensional (2D) surfaces, making it challenging for relevant analyte molecules to ‘collide’ with them at practically relevant time scales. This is especially true when the concentration is low, typically below about picomolar level. In one approach to overcome this issue, antibodies attached to magnetic particles are directed to sorting wells under a magnetic field to detect protein molecules at subfemtomolar concentrations17. Other suggested approaches include a multi-length scale electrode architecture, where the surfaces of micro and mesoscale structures enable an active interaction of the analyte molecules even at low concentrations, while nanostructures on the electrode surface give the surface enhancement that can lower the limit-of-detection (LoD) for sensing in a reasonable amount of time14.Electrochemical transduction is a well-established surface-sensitive technique to detect biomolecules of various types18) was used to create 3D complex device architectures with solid truss members leading to aspect ratios >20:119. The 3D printed 3D geometry enables an electrode architecture that has multiple hierarchical length-scales . Another feature of 3D printing is that different material types can be seamlessly integrated with the complex architecture being printed. For example, atomically thin nanomaterials such as graphene flakes can be combined with the electrode to enable ultra-high surface area in addition to the micro-patterning achieved by printing. Lastly, since the printed microarchitectures are typically formed on a substrate19, they can be combined with microfluidic devices where analyte molecules can be conveniently introduced into the fluid flow of the device.The emergence of additive manufacturing, i.e., three-dimensional (3D) printing, has allowed the fabrication of micro-to-mesoscale multi-level 3D device architectures that were difficult, if not impossible, to build via conventional lithography. This was made possible via the recent work in our group where Aerosol Jet (AJ) nanoparticle printing , dementia with Lewy bodies, and Huntington’s disease, while its high level is associated with brain cancers such as neuroblastoma22. Though the physiological range of dopamine in human biofluids  are different, according to Human Metabolome Database, the dopamine concentration in blood is <130 pm (see Supplementary Table 23. Prior reports on the detection of dopamine at nanomolar (nm) concentration do exist25. For example, an electrochemically grown reduced graphene oxide (rGO)-based electrode has been utilized to detect dopamine at 0.1 µm via direct catalytic oxidation reaction26. All these methods involve nanomaterials on 2D surfaces to increase the detection sensitivity. Other dopamine detection methods include enzyme-linked immunosorbent assays (ELISA) (~1.26 nm)27, high-performance liquid chromatography (~65.3 pm)28, ultraviolet spectroscopy (~1 μm)29, fluorometry assays (~20 nm)30, electrochemiluminescent (100 nm)31, 2D-material-based electrochemical sensors (50 pm)20, porphyrin-based metal–organic framework nanocrystals coated poly (40 nm)32, and enzyme-based electrochemical sensors (1 nm)34.Dopamine is one of the biomolecules that can be detected at low concentrations with significant clinical advantages1, that overcomes the limits of the current electrode architectures to achieve femtomolar level of LoD. The focus is on creating a multi-length-scale electrode architecture consisting of AJ printed micro- and mesoscale hollow silver pillars that ‘protrude’ into the microfluidic channel containing the analyte, dopamine, and, coating the electrodes with atomically thin sheets of rGO. The multi-scale architecture is expected to accelerate the electrochemical redox reaction by allowing a more effective interaction of the analyte molecules  with the atomically thin rGO to achieve a sub-picomolar LoD. By nanostructuring the surface and controlling multi-length-scale electrode structure, the LoD for dopamine was tuned from micromolar to femtomolar concentration, a significant result for clinically relevant diseases. This construction is also designed to enable an enzyme-free detection of dopamine with a wide linear measurement range (fm to nm range). Lastly, the architecture is expected to facilitate the introduction of perfluorinated polymer layer, or Nafion, into the electrode, enhancing the selectivity of the sensor by eliminating negatively charged interfering molecules.In this work, we are motivated by the exciting possibility of creating a nano-to-mesoscale biosensing platform, envisioned in the theoretical work in literatureWe first describe the AJ nanoparticle printing method used to fabricate the multi-length-scale biosensing platform developed in this work. Figure 35. We decided to explore sintered silver microelectrodes decorated with rGO nanoflakes for the sensing platform because of the excellent electro-catalytic properties demonstrated by these materials in previously reported literature36.Figure 1. As shown in Fig. Figure ® software. Figure 37. Unlike the planar electrodes, the 3D cylindrical electrodes used in this work show a uniform radial diffusion in all directions combined with linear diffusion 6]3−/4−) redox mediator  via electrochemical oxidation during measurements, potentially reducing the current. In the current work, however, it is expected that the protonated Nafion and multiple layers of rGO covering the silver electrodes prevent the Cl− ions from reacting with silver. To verify this, we measured the effect of Cl− concentration on a base electrode made of silver nanoparticles as shown in Fig. − concentration while the reduction has a minute change (2 µA). In fact, for the physiological range of Cl− concentration in human serum (96–106 mm), the change in current is insignificant. We thus sense dopamine by taking advantage of the fixed oxidation potential of our 3D printed device.Cyclic voltammetry (CV) studies were carried out to investigate the electrochemical behavior of 2D silver and 3D silver micropillar geometries coated with rGO as shown in Fig. tor Fig. . The CV tor Fig. . CompareRct) at the electrode-electrolyte interfaces. Figure Rct estimated from these plots , respectively. The Rct and the double-layer capacitance (Cdl) are obtained from the real (Zre) and imaginary (Zim) parts of complex impedance at different frequencies for a parallel RC circuit given by39,Rs is the solution resistance. The heterogeneous electron transfer (HET) rate constant (k0) for all the electrode geometries is defined39 asT is the temperature (298 K), R is the gas constant (8.314 J/K mol), n is the number of electrons that are transferred in the redox couple (1), F is Faraday’s constant (96485.3 C/mol), A is the area of the electrode, and C0 is the bulk concentration of the redox species (1 mm). Interestingly, the dramatic reduction of Rct for 3D Ag/rGO micropillar electrodes could be due to both the linear and spherical diffusion of electrons surrounding the 3D geometry of micropillars from bulk electrolyte, leading to a higher electron transfer rate (k0) compared to 2D Ag/rGO electrode  for both 3D Ag/rGO and 2D Ag/rGO electrodes were evaluated. The value of D for the AJ printed 3D silver micropillar electrodes was higher than that for planar geometries (Supplementary Table 6]3−/4− ions (or electrons) to the current collector. In addition, in the presence of low concentration of dopamine molecules, the 3D geometry will enhance the interaction of the analyte molecules with the electrode. We thus confirm that the sensing platform developed in this work enhances reactivity of the redox species as predicted in the COMSOL simulations in Fig. To obtain a deeper insight into the advantages offered by the AJ printed 3D Ag/rGO electrode geometry, we also carried out electrochemical impedance spectroscopy (EIS) at a frequency range of 1–10,000 Hz to investigate the charge transfer resistances  and dopamine (0.1 µm)  and two protons (2H+)41. The biochemical reaction of dopamine molecules is shown in the inset of Fig. 43. As the oxidation potential (−0.14 V) is selective to dopamine only (w. r. t. ascorbic acid), we chose this potential (−0.14 V) for the sensing of dopamine in the next experiments.Once we established the enhanced reactivity of the redox species in the 3D geometry, the 3D Ag/rGO micropillar sensor was tested using CV technique in presence of ascorbic acid (0.1 µµm) Fig.  at the sOnce the oxidation potential for the enzyme-free dopamine detection was obtained, chronoamperometric studies were carried out for the 2D Ag/rGO (0 × 0 array), 3D Ag/rGO (4 × 4 array), and 3D Ag/rGO (10 × 10 array) sensors. Before dopamine sensing, the baseline of three sensors was obtained using chronoamperometric method at a fixed potential of −0.14 V  under a diffusion control process and is given by,n is the number of electrons, A is the electrode area, F is the Faraday constant, D0 is the diffusion co-efficient of the redox species, t is the time, and C0 is the bulk concentration of the redox species. Although the current paper focuses on in vitro sensing of dopamine, we depict the dopamine transfer through the synaptic cleft between pre-synaptic neuron to post-synaptic neuron via ligand-gated ion channels as presented in Fig. The dopamine sensing was based on the principle of chronoamperometric method wherein the current of the micropillar array electrode was measured at a fixed sensing potential with respect to the RE in presence of a variety of electrolyte solutions. The resulting current at the WE due to the faradaic process for a fixed potential step was recorded as a function of time. During sensing of dopamine, the fixed potential was stepped to a value where the dopamine showed a redox behavior. According to the Cottrell equation, the current decays with the square root of time . The sensor was washed using pbs solution before exposing a new solution of dopamine for the next chronoamperometric measurements. As the dopamine concentration increased, it was observed that the output current varied for both sensors. This change is attributed to catalytic oxidation of dopamine resulting in the formation of dopamine-o-quinone, and two electrons , 3D (4 × 4), and 2D (0 × 0) are measured as 41.7:15.5:1 . Similarly, the 3D sensor with 4 × 4 array also showed two linear response regions for dopamine concentration , and 10 to 1000 μm (r2 = 0.97) concentration of dopamine. However, the 2D sensor with 0 × 0 array showed only one linear response region  concentration of dopamine. Their analytical sensitivities are found to be 1 μm (0 × 0), 10 pm (4 × 4), and 10 fm (10 × 10). The slope values are found to be higher in the case of 3D sensor with 10 × 10 geometry. LoD is determined for all three sensors by a method reported in literature47, and calculation is shown in Supplementary Section m and a wide detection range of 10 fm–10 μm for dopamine compared to other sensor configurations such as 4 × 4 array (0.87 pm) and 0 × 0 array (11.2 µm). In addition, the analytical sensitivities are found to be 1 µm, 1 pm and 10 fm for the three sensor configurations of 2D Ag/rGO (0 × 0 array), 3D Ag/rGO (4 × 4 array), and 3D Ag/rGO (10 × 10 array), respectively. These results clearly demonstrate the superior performance obtained by using 3D architectures for the electrodes in the electrochemical cell.The 3D Ag/rGO sensor with 10 × 10 array configuration showed two linear responses for dopamine; first response is from 10 f48, nano-gold49, planar carbon paste50, 3D carbon51, 2D plasmonic hole array52, nickel oxide nanoparticles34, and planar gold (Au)-CoP53, the use of 3D Ag/rGO micropillar geometry with its nano-to-meso scale architecture improved the LoD for dopamine detection by about nine orders of magnitude. As the measured physical range of dopamine in human blood and plasma is as low as 0.01–0.48 nm, and 0.13 nm, respectively, the method described in this paper demonstrates not only a clear improvement over the current state-of-the-art, but also a highly useful method for neurophysiological applications.Table 34. Figure , and 1000 μm concentrations. Without dopamine, the 3D and 2D sensor signals in fbs and rs solutions are found to be very close in comparison to only pbs solution as evident by their low relative standard deviations (RSDs) of ±5.01% (3D) and ±6.12% (2D), respectively, indicating that the sensor does not show any response to a high content of proteins such as albumin or other molecules present in the serum fluids  sensor, on the addition of 100 μion Fig. . Furtherion Fig. . As the m concentration. The results are summarized in Fig. n = 18) from the mean signal (47.6 µA). Once dopamine is added into the diluted HP, the sensing current is increased significantly compared to the baseline signal . With the spiked-plasma dilution, the sensor showed a deviation of ±3.8 µA from the mean signal (88.8 µA), which is ~ ±4%. Though the concentration of HP is high, the other neurotransmitters or proteins in plasma do not affect the dopamine sensing signal. This may be due to the presence of Nafion on the sensor surface which repels negatively charged proteins in plasma. Supplementary Fig. To establish the clinical relevance of the biosensing platform proposed in this work, we also conducted dopamine sensing in human plasma (HP) and artificial serum (AS) using the 3D 10 × 10) sensor. We diluted these body fluids in ratios of 1:5 to 1:500 (HP/AS:pbs) and spiked them with dopamine at 1 p × 10 senm. For this test, we used two different concentrations of dopamine, 100 µm for 2D Ag/rGO sensor and 1 nm for 3D Ag/rGO sensor. The 2D Ag/rGO sensor  determines the selectivity where the large area of 3D electrode structure allows a catalytic reaction only for the dopamine molecules. A low RSD of ±3.4% of 3D Ag/rGO sensor indicates the high specificity with the interfering molecules compared to the 2D Ag/rGO sensor (RSD~ ±6.4%). Thus, compared to the 2D sensor, the AJ printed 3D Ag/rGO micropillar electrodes provide a high selectivity for dopamine against other similar interfering neurotransmitters.The selectivity test of the sensors was conducted using chronoamperometric method with and without dopamine in presence of a variety of interferents such as ascorbic acid (AA), histamine (His), uric acid (UA), epinephrine (EP), and 3,4-Dihydroxyphenylacetic acid (DOPAC) at a potential of −0.1 V Fig. . For thesor Fig.  exhibitesor Fig.  exhibitem and 1 pm at −0.14 V for 2D Ag/rGO and 3D Ag/rGO sensors, respectively. The high uniformity of 3D micropillar geometry  C 1s, (c) O 1s, (d) Na 1s, (e) S 2p, and (f) P 2p peaks. Peak intensities have been rescaled to show additional details, while quantified results are shown in Supplementary Table d3/2 (374.3 eV) and Ag 3d5/2 (368.3 eV) did not shift and no new peaks appeared at higher binding energies, meaning that the oxidation state of Ag did not change after dopamine sensing . Interestingly, a trace amount of sulfur was detected in as-fabricated micropillars but disappeared after dopamine sensing  that addresses issues such as biofouling. For example, in this paper, the use of silver allows direct oxidation of dopamine due to its electro-catalytic properties. Further, it is noted that 3D printing can create structures that have imperfections larger than conventional lithography. For example, the micropillar structures used in the current work , high sensitivity, and in vitro capability of sensing dopamine in spiked human plasma and serum samples. This work, in addition to being an important advance in biosensing enabled by advances in microelectronics fabrication, enables the detection of dopamine at hitherto undetectable ranges, which will open new pathways for the detection and treatment of neurodegenerative and other diseases.57. The rGO nanoflakes in powder form were purchased (CAS‐No. 7782‐42‐5) from ACS Materials LLC, Pasadena, CA, USA. As per the manufacturer, the rGO was synthesized from graphene precursor via a reduction process of hydrazine (N2H4) treatment, and the rGO sheets has a thickness of ~1 nm, a conductivity of >500 Sm−1, and a diameter of 0.5–10 µm. The solvents used for this ink were deionized (DI) water and ethylene glycol. Ethylene glycol acted as a humectant which is known to help in the formation of non-planar structures required for this work19. Fetal bovine serum (fbs) and rabbit serum (rs) were obtained from Sigma Aldrich . Polydimethylsiloxane or PDMS  was used as the housing material to form the microfluidic channel in the sensor device by mixing the base to hardener at a ratio of 10:1 using a process described in the next section. Artificial serum (SMx Serum) was purchased from UTAK, Inc., Valencia, CA, and HP (product number: P9523) was obtained from Sigma Aldrich . The experiments with biological fluids were conducted in the biosafety cabinet level 2 (BSL 2) facility in the biomedical engineering department at Carnegie Mellon University.To construct the 3D hollow micropillar array, we used silver nanoparticle ink . The size of the nanoparticles in the ink was 30–50 nm, the ink viscosity was about 1.5 cP, and the silver particle loading in the ink was about 40 ± 2 wt%. Silver was chosen because of the excellent electro-catalytic properties exhibited by silver nanoparticles in oxidizing other biomolecules such as glucose® tape . This pattern was used to create three electrodes, namely, WE, CE, and RE. The AJ printing process was then used to print 3D micropillar array (10 × 10 hollow pillars over an area of 2 mm × 2 mm) on the surface of the WE . Inside the nozzle, the droplets were focused towards the nozzle tip with the help of a sheath gas  to form a micro-jet. The process of printing was carried out by a continuous flow of droplets which was diverted or resumed by the movement of a shutter (not shown). The diameter of the nozzle tip was 150 μm, which is known to give rise to an aerosol stream having a diameter of ~10–15 μm. Before printing, the geometry of the conductive part was drawn in AutoCAD, 2020 using a program in the software AutoLISP  and converted to a ‘prg’ file compatible with the printer software. The desired shape of an individual hollow micropillar is shown in Fig. The Aerosol Jet 3D printer  used in this work had three atomizers (two ultrasonic atomizers and one pneumatic atomizer), a programmable heated X-Y motion stage (i.e. platen), and a deposition head. For the construction of the microelectrode array in this work, an ultrasonic atomizer was used to aerosolize the silver nanoparticle ink as shown in Fig. Figure −1), added with Nafion (5.0%) solution  and sonicated for 1 h. A PDMS fence was created that surrounded the silver microelectrode arrays, and a 20 µL solution of the mixture of rGO and Nafion was drop-casted onto the array surface and dried at 85 °C for 2 h. This process was repeated three times to ensure a uniform coverage by rGO flakes of the microelectrodes array. Similarly, other electrodes such as 2D (0 × 0 array) and 3D (4 × 4 array) were functionalized with rGO flakes and Nafion. Adding the perfluorinated polymer layer of Nafion onto microelectrode array can act as a barrier and repels the negatively charged neurotransmitters such as ascorbic acid (and other negatively charged neurotransmitters) but allows the interaction with positively charged molecules such as dopamine58, thereby providing high selectivity. Note that based on the 3D construction of the electrode alone, the area increases for the micropillar structures over a planar electrode  projected to sample surface. Further, 150 eV pass energy and 400 ms dwell time were used for the XPS measurement. The signal was detected from top of the pillars of the array.The micropillar array electrodes were characterized using scanning electron microscope or SEM . The surface chemistry of the samples with silver (without rGO) before and after electrochemical measurements was assessed by XPS using a Thetaprobe . The XPS characterization was performed at room temperature with a base pressure of 3 × 10−). At the electrode boundary, the species ‘B’ oxidizes to ‘A’ wherein the bulk concentration is uniform, and the product concentration is zero. The domain equation in this simulation was the diffusion equation of Fick’s second law. To evaluate the total current of the micropillar arrays, we used the electroanalytical Butler-Volmer equation59 for the oxidation reaction. In this simulation, a total of eight array configurations, namely, 0 × 0, 1 × 1, 2 × 2, 4 × 4, 6 × 6, 8 × × 8, 10 × 10, 12 × 12, 14 × 14, and 20 × 20, were analyzed. The diffusion profiles of the species at the electrode and the total current were calculated to obtain an optimum array configuration for the sensor.Simulations were carried out to investigate the diffusion profiles and the total current inside the microfluidic chamber during the oxidation/reduction of dopamine for different micropillar array configurations using COMSOL Multiphysics software . In the electroanalysis module of COMSOL, electrochemical reaction was used for a single species (B) and formed product (A) via oxidation (B + ↔A + eFor statistical analysis, the OriginPro 2020 and GraphPad Prism 6.02 were used. Solidworks 2019 was used to create 3D schematics. AutoCAD 2020 was used to create circular patterns for 3D printing. The software associated with the Aerosol Jet 3D printer is the Kewa Gadget, wherein different control managers, namely, motion manager 3.2.83, process control 3.2.83, and vision manager 3.2.83 were used to control the 3D printing of nanoparticles.Further information on research design is available in the Supplementary InformationReporting Summary


Neurotransmitters are biochemical molecules that transmit a signal from a neuron across the synapse to a target cell, thus being essential to the function of the central and peripheral nervous system. Dopamine is one of the most important catecholamine neurotransmitters since it is involved in many functions of the human central nervous system, including motor control, reward, or reinforcement. It is of utmost importance to quantify the amount of dopamine since abnormal levels can cause a variety of medical and behavioral problems. For instance, Parkinson’s disease is partially caused by the death of dopamine-secreting neurons. To date, various methods have been developed to measure dopamine levels, and electrochemical biosensing seems to be the most viable due to its robustness, selectivity, sensitivity, and the possibility to achieve real-time measurements. Even if the electrochemical detection is not facile due to the presence of electroactive interfering species with similar redox potentials in real biological samples, numerous strategies have been employed to resolve this issue. The objective of this paper is to review the materials  that are frequently used for the electrochemical biosensing of dopamine and point out their respective advantages and drawbacks. Different types of dopamine biosensors, including (micro)electrodes, biosensing platforms, or field-effect transistors, are also described. Dopamine is a neurotransmitter playing essential roles in the cardiovascular and central nervous systems. As such, high dopamine levels indicate cardiotoxicity leading to rapid heart rates, hypertension, and heart failure . On the Moreover, electrochemical biosensors generate a fast and sensitive response, allow monitoring in real-time, and can be miniaturized enough to be implanted in living cells. However, several issues need to be resolved. First, biosensors must be able to give a sensitive response in the appropriate range of concentration . Second, the biosensors must be selective enough to discriminate dopamine from its interferents, such as ascorbic acid or uric acid, which undergo oxidation within the same potential window as dopamine.The selectivity of the dopamine biosensor is especially important and challenging when studying real samples (human serum or blood) due to the complexity of the biological samples. Indeed, such matrix does not contain only commonly occurring interferents such as ascorbic acid or uric acid, but also other neurotransmitters and molecules. To perform electrochemical detection of dopamine in real samples, it is generally necessary to add a few steps to sample preparation to reduce the matrix effect ,6. For bTo overcome these difficulties, researchers are developing new sensing materials that can be deposited onto the surface of an electrode. These can, in turn, increase the electrocatalytic properties of the electrode to obtain a more sensitive and selective response to dopamine. The most popular strategies used for the modification of electrodes are: the deposition of nanomaterials acting as redox mediators; the deposition of compounds containing specific groups that can facilitate the charge transfer; the deposition of nanostructures with a high specific surface area to increase the sensitivity of the electrode.The main goal of the current review is to summarize the recent studies of nanomaterial-modified electrodes that were proven to be effective for the electrochemical detection of dopamine with high sensitivity and in a selective manner. Of the various nanomaterials available, we focused on three classes of materials, i.e., metal and metal oxide nanomaterials, carbon nanomaterials (graphene and carbon nanotubes), and polymers (conducting polymers and molecularly imprinted polymers), which can all improve the electrocatalytic oxidation of dopamine, as briefly described in When target biomolecules are captured by a sensing material deposited on the working electrode of an electrochemical biosensor, an analytical measurable signal is generated A. In theIn amperometric biosensors, the current produced during the oxidation or reduction of an electroactive biological element at a constant potential applied between a working electrode and a reference electrode is measured, providing specific quantitative analytical information ,10,11. TDuring cyclic voltammetry experiments, a current is produced by sweeping the potential applied between two electrodes over a range that is associated with the redox reaction of the analyte. This redox reaction generates a change in peak current that can be correlated to the concentration of the analyte, thus leading to specific quantitative analytical information ,16,17. TFor example, the oxidation of dopamine using cyclic voltammetry leads to an oxidation peak, which is characteristic of this biomolecule. By studying the evolution of the intensity of the oxidation peak present in the cyclic voltammograms for different dopamine concentrations, it is possible to draw calibration curves and quantify this compound C,D.Fast-scan cyclic voltammetry (FSCV) is a derivative technique that can be used to address analytical challenges arising from biomedical needs to measure locally low concentrations of neurotransmitters ,19. WithThe monitoring of dopamine by FSCV has been exhaustively described by Venton and Cao . It consDifferential pulse voltammetry is a derivative of the linear sweep voltammetry technique with a series of regular voltage pulses superimposed on the potential linear sweep ,26,27. INeurotransmitter biosensors and neural-electrical interface devices are being more and more developed for monitoring the release of neurotransmitters in vivo and restoring or supplementing the function of the nervous system, respectively. For this purpose, microelectrodes are of great interest to monitor locally the fluctuations of the neurotransmitter concentrations ,28 and rMetal electrodes are used due to their high electrical conductivity and to the ease of fabrication of metal microelectrode arrays using microsystems technology . In partHowever, carbon-fiber electrodes are the most widely used for the electrochemical characterization of dopamine oxidation ,41 becauIn addition, carbon-fiber electrodes can be functionalized or modified in order to increase their selectivity for cationic dopamine over anionic interferents such as ascorbic acid. Thus, a Nafion coating can be deposited on carbon electrodes to repel negative interferents since Nafion is an anionic polymer . SimilarCarbon-fiber microelectrodes are also used to perform single-cell amperometry measurements, which allows monitoring the release of electroactive molecules, such as dopamine from individual vesicles, since they can be positioned in the close proximity of a cell thanks to their small size ,54. As aMetal nanoparticles (NPs) are an advantageous material for biosensing  owing toThus, a nanostructured gold surface consisting of closely packed outwardly growing spikes showed a significant electrocatalytic effect for the electrooxidation of dopamine due to the presence of numerous surface-active sites . The resRecently, another dopamine biosensor based on 3D nanocone-shaped gold nanostructures was fabricated to detect dopamine from neuronally differentiated rat phaeochromocytoma (PC12) cells. Cyclic voltammetry confirmed that the gold nanoelectrodes showed higher oxidation peaks towards dopamine than the bare nanoelectrode . FurtherPalladium nanoparticles were also used to modify electrodes, which displayed good electrochemical catalytic activities towards dopamine since the calibration curve obtained using differential pulse voltammetry was in the range of 0.5–160 μM, and the detection limit was 0.2 µM . Some doAlthough metal electrodes have many advantages, they could suffer from a lack of selectivity. To address this issue and enhance selectivity, a strategy consists of incorporating aptamers, which are well-known small oligonucleotides capable of binding to a specific target of interest, can be employed. Thus, by combining the high conductivity and specific surface area of metal nanoparticles with the selectivity of aptamers, it is possible to obtain very efficient biosensors. An electrode modified with gold nanostars and a dopamine-binding aptamer exhibited a very low detection limit of 0.0019 nM. The presence of many interferents did not hinder the sensitivity of the biosensor due to the specific binding between the aptamers and the dopamine molecules . Similar3O4 nanoparticles, which electrocatalyzed the oxidation and increased the sensing of dopamine, leading to a linear range of 0.1–90 µM and a detection limit of 1 nM [+ (extracellular K+ is very frequently used to induce a release of positively charged dopamine molecules by living cells). Another electrochemical sensor based on NiO-lacy flower-like geometrical structure with semi-spherical head surfaces associated with abundant and well-dispersed tubular branches with needle-like open ends was developed [Metal oxide nanoparticles and nanostructures can also be used for electrochemical sensing of dopamine owing to their high surface area and good biocompatibility . Thus, i of 1 nM   when compared with bare electrode [2 led to a wide linear response range (0.006–181 μM) and a very low detection limit of 2 nM. Another photoelectrochemical sensor, including a nanoMoS2-modified gold electrode, displayed a very high sensitivity with a limit of detection of 2.3 pM in the presence of other interfering molecules, such as ascorbic acid, uric acid, and cysteine [A flake-like MoSlectrode . The flacysteine . Hierarccysteine .3O4 materials were synthesized by using mesoporous silica as a hard template [3O4 with high surface area modified glassy carbon electrode showed a high catalytic activity to dopamine with a detection limit of 0.8 nM and a linear range between 2 and 600 nm. The dopamine released from cultured PC12 cells was also measured in real-time using amperometry. Similarly, highly ordered mesoporous ZnFe2O4 was prepared via a nanocasting method and was found to be highly sensitive in the electrochemical detection of dopamine in a wide linear range from 2 to 600 nM and a low detection limit of 0.4 nM [+ stimulation of living PC12 cells in a neurological environment.Highly ordered mesoporous Fetemplate . The ordf 0.4 nM . It was Some examples were given here describing dopamine biosensors based on metal oxide nanostructures. However, if metal oxides have the advantages of high surface area and biocompatibility, they have the disadvantage of low conductivity. That is why they are often combined with more conducting materials, in particular carbon materials, to obtain more efficient biosensing of dopamine, as it will be shown in subsequent sections.Carbon materials are used in various fields due to their thermal stability, chemical resistance, and excellent mechanical properties ,79,80. TCarbon nanotubes (CNTs) are grouped into two sorts: cylinder-shaped single-walled CNTs (SWCNTs) with diameters in the range of a nanometer, and multi-walled CNTs (MWCNTs), consisting of nested single-wall CNTs weakly bound together by van der Waals interactions. Individual CNT walls can be metallic or semiconducting depending on the alignment of the graphene lattice with respect to the tube axis. CNTs have been widely used as electrode modifiers in electrochemical biosensing  and showNCTs are frequently used to decrease the oxidation potential of dopamine and facilitate its biosensing. For example, SWCNTs and MWCNTs were introduced in a carbon electrode modified with 5-amino-3′,4′-dimethyl -biphenyl-2-ol. The oxidation of dopamine occurred at a potential about 170 mV (resp. 160 mV) less positive than that of the electrode, which did not contain CNTs, thus leading to a linear response over a wide concentration of dopamine  ,87 Tabl.It is also possible to incorporate negatively charged SWCNTs into carbon electrodes. For example, negatively charged SWCNTs, obtained by the introduction of carbonyl functionalities, selectively attracted the cationic dopamine towards the electrode and repelled the anionic ascorbate and uric acid coexisting in the same solution (detection limit: 15 nM) . SimilarSWCNTs can also be combined with Nafion and poly(3-methylthiophene) to obtain synergetic effects since Nafion has high antifouling capacity and permeability to cations when SWCNTs and conducting polymers have high electrocatalytic properties. The resulting electrode led to the oxidation of dopamine, uric acid and ascorbic acid at distinguishable potentials to form well-defined and sharp peaks, making possible simultaneous determination of each species. Moreover, the proposed electrode was advantageously employed for the determination of dopamine in real pharmaceutical and clinical formulations . Another2-decorated cobalt oxide polyhedrons were synthesized. The resulting composites possessed good porosity, large electrochemical area, roughened surface, and excellent electrocatalytic ability as proven by the limit of detection (13 nM) of the dopamine biosensor, which was successfully tested in rat brain and human blood serum samples [MWCNTs can also be modified with nanoparticles. Thus, a biosensor allowing the monitoring of extracellular dopamine in neuronal cells was developed, combining MWCNTs and Ag–Au nanoparticles in the same electrode. The presence of metal nanoparticles increased the electron transfer rate and the sensing performances of the sensor, leading to a linear range of 3 nM–2.3 µM and a detection limit of 0.23 nM . Similar samples .2 network of carbon atoms. Graphene and its derivatives graphene oxide (GO) and reduced graphene oxide (rGO) have emerged as promising materials for wide practical applications, including electrocatalysis and electroanalysis, due to their beneficial characteristics, including high thermal and electrical conductivity [Graphene is a 2D nanomaterial consisting of a single layer of spuctivity , excelleuctivity , a wide uctivity . In addiuctivity .The addition of graphene or (reduced) graphene oxide is sufficient to observe an increase in the response of dopamine sensors and to obtain a wide linear range and low detection limit: 10 nM–100 µM and 1 nM for graphene using cyclic voltammetry , and 1–8Poly (PEDOT) is an excellent conducting polymer for electrochemical detection of dopamine due to its low oxidation potential, and it can be easily deposited onto graphene. Such modified electrodes provided a low detection limit of 0.33 µM and an excellent peak separation between dopamine and its interferents . Polypyr3O4-graphene nanospheres coated with Nafion exhibited excellent electrocatalytic activity towards the oxidation of dopamine [2O–rGO nanocomposite was used for dopamine determination by cyclic voltammetry [3O4 [Metal oxides are also frequently combined with graphene to obtain modified electrodes. Thus, the carbon electrodes modified with core–shell Fedopamine . Under ttammetry . The restry [3O4  or NiO [try [3O4 , have alA graphene–nickel hydroxide modified carbon electrode exhibited an appreciable electrocatalytic effect for the simultaneous detection of lower concentrations of dopamine compared to graphene modified carbon electrode . The det−16 M and selectivity among interfering molecules. In another study, the rGo composite was modified with dendritic platinum nanoparticles, which increased the sensitivity of sensing dopamine released from pheochromocytoma cells  [Metal nanoparticles can be combined with graphene and enhance its electrocatalytic properties. Thus, an electrochemical sensor was prepared by modifying an electrode with rGO and gold nanoparticles having great conductivity and a large surface area . The res–100 μm) , the bioConducting polymers are very common modifiers that can be chemically or electrochemically deposited over bare electrodes from their monomer solutions. They have an extended π-conjugated structure with alternating single and double bonds across the polymeric chain, which causes delocalization of the electrons in the polymeric backbone, and is responsible for their outstanding electrical and optical properties. In particular, conducting polymers can reach excellent electrical conductivity comparable to the one displayed by metals. In addition, they present other advantages for use in electrochemical biosensing, such as the possibility to be easily modified with functional groups or with nanoparticles, such as metal oxide nanoparticles  and metaPolypyrrole is one of the most widely used polymers for biosensing due to its good redox activity, water solubility, and biocompatibility, as well as ease of modification and ability to form nanostructures. Thus, polypyrrole nanofibers were deposited on modified electrodes by electropolymerization. The resulting sensor showed high selectivity to dopamine with a low detection limit of 7 nM thanks to the high reactivity due to the large surface area of the nanofibers  Table 5Table 5. + concentration. A comparison between the generated current on bare gold electrodes and polypyrrole-modified electrodes illustrated the clear advantage of the modification, yielding 2.6-fold signal amplification. It is also possible to incorporate a mediator such as a ferrocene in a conducting polymer film, such as PEDOT, to enhance the electron transfer between the modified electrode and dopamine biomolecules, hence facilitating the biosensing of the neurotransmitter. The resulting biosensor presented a linear range between 0.01 and 0.9 mM, and a limit of detection of 1 µM [Another biosensor using electropolymerized poly (2-napthol) orange film deposited on a carbon electrode is worth considering due to the importance of charges and interactions in the sensing mechanism . Indeed, of 1 µM .Several works have been dedicated to the use of electrodes modified by a conducting polymer and gold nanoparticles to detect dopamine. For example, an electrode was modified by gold nanoparticles, poly and sodium dodecyl sulfate . This mo+ stimulation.Composites were also prepared from conducting polymers and carbon nanomaterials. For example, a nanocomposite composed of poly  doped with graphene oxide was electrodeposited on an electrode and exhibited lowered electrochemical impedance and excellent electrocatalytic activity towards the oxidation of dopamine  due to the large surface area, which provided many active sites that accelerate the electron transfer process of dopamine . An elec2+, K+, Na+, glucose, urea, sucrose, citric acid, and cystine, and the sensor was successfully utilized in pharmaceutical samples.Several biosensors based on conducting polymers and carbon nanotubes have been developed. For example, electrodeposited poly doped with pure CNTs were deposited on a carbon paste electrode leading to a sensitive dopamine biosensor with excellent catalytic property, a linear range from 0.1 to 20 μM, and a detection limit of 20 nM . SimilarThe field-effect transistor (FET) is a type of transistor that uses an electrical field to control the flow of current. The electrical signals of a FET are generally expressed as the changes in current intensities. FET is currently one of the most popular types of devices in the biosensor field due to its extreme sensitivity ,145. Sin4 aqueous solutions in order to induce a covalent bonding between the Pt4+ ions and the negative charge of the carboxylate group present in the polymer structure. Then, by the addition of NaBH4, Pt4+ ions were reduced to Pt nanoparticles. The resulting hybrid particles were then immobilized on an amine-functionalized interdigitated-array electrode substrate through the formation of covalent bonds with amine groups. The resulting Pt–Polymer-based FET biosensors exhibited high sensitivity and selectivity towards dopamine at unprecedentedly low concentrations (0.1 pM). Finally, a potentiometric FET sensor whose sensing element resides at the Au gate–aqueous solution interface by means of a self-assembled monolayer composed of cysteamine and 4-formylphenyl boronic acid was fabricated [Another FET was functionalized with carboxylated polypyrrole nanotubes/aptamer and used to detect the dopamine secreted by living cells . A very bricated . Even ifBorrowing inspiration from the high affinity and specificity of biorecognition probes, such as antibodies and aptamers towards their target molecules, molecular imprinting technique has been developed to prepare a molecularly imprinted polymers (MIPs) with the purpose of acting as synthetic receptors for a targeted molecule . Indeed,−14 M.Using this strategy, an electrochemical sensor was developed by the electropolymerization of conducting pyrrole onto a silica colloidal crystal template modified glassy carbon electrode surface in the presence of dopamine  Table 6Table 6. Conducting polymer nanowires also offers great potential in the preparation of biosensors due to their large surface area and electrical conductivity. That is why 3D structures comprising polypyrrole nanowires and MIPs, obtained by electropolymerization of dopamine with o-phenylenediamine via cyclic voltammetry technique, were prepared by electropolymerization on the surfaces of a glassy carbon electrode . The res−11–5.0 × 10−6 M and the low limit of detection of 10 pM, which could be due to the high number of cavities for binding dopamine by the π–π stacking between the aromatic rings and by the hydrogen bonds between the amino groups of dopamine and the oxygen-containing groups of the polymer.As previously presented, gold and carbon nanomaterials can also be combined with MIPs to increase the conductivity of these sensing materials. Thus, a dopamine-imprinted sensor in which gold nanoparticles were electrodeposited onto glassy carbon electrode, followed by self-assembly of the monomer p-thioaniline monolayer onto the electrode surface, through Au–S bonds formed between gold nanoparticles and thiol groups . The amiThe detection of dopamine in real-life applications is a promising and exciting area of research, as indicated by hundreds of publications on this topic. Electrochemical biosensors are a promising technique for the detection of dopamine or other neurotransmitters. In this review, we summarized recent studies of nanomaterial-modified electrodes for the detection of dopamine. In recent years, most of the studies were devoted to the preparation of new sensing materials to improve biosensor performances. Thus, various nanomaterials  were reported to be excellent in improving both the sensitivity and the selectivity of modified electrodes. Specifically, in the case of electrochemical detection, the nanomaterials showed their ability to facilitate electron transfer reactions on the electrode, improving the detection of unique oxidation peaks or significant currents.Now, modified electrodes have proved that they can be useful for dopamine detection in standard conditions and exhibited detection limits in the picomolar range, as well as selectivity towards dopamine even in the presence of interferents. It can be considered that they have shown a grade of maturity good enough to implement them in real analytical applications. Thus, the technology must now move from standard conditions to biomedical and clinical applications and be implanted in point-of-care devices. The future of dopamine biosensors probably consists of the development of miniaturized systems or platforms (consisting of arrays of nanoelectrode sensors or field-effect transistors) that must be portable, non-invasive, cheap, wearable, and capable of reducing the time and frequency of sampling. Thanks to the recent advances in miniaturized system technologies, it can be expected that future electrochemical biosensors will be capable of performing localized measurements of dopamine levels from living neurons that will facilitate the discovery of new types of drugs and techniques, which will ultimately contribute to the treatment of various dopamine-related diseases, including Parkinson’s disease, schizophrenia, Alzheimer’s disease, stress, and depression.


Staphylococcus aureus teaches us that once AMR bacteria have established their high endemicity, controlling such AMR bacteria requires time and is challenging. On the other hand, the epidemiology that multidrug-resistant Acinetobacter sp. exhibits when a strict containment policy for AMR bacteria was introduced in the early phase of its emergence and spread reveals that it is possible to control it. Detailed epidemiology provided by these two different national surveillance systems in Japan enabled us to set up the goal for controlling each AMR bacteria at the hospital level to the prefecture/national level. It is the public health authorities’ responsibility to maintain a good surveillance system for AMR bacteria and share the data and findings with healthcare professionals and academicians.The Ministry of Health, Labour and Welfare (MHLW) of Japan has conducted two national surveillance systems for approximately 20 years to monitor antimicrobial resistance (AMR) in bacteria: the National Epidemiological Surveillance of Infectious Diseases (NESID) and the Japan Nosocomial Infections Surveillance (JANIS). Data accumulated for 20 years by these two surveillance systems have helped depict the epidemiology of the representative AMR bacteria in Japan chronologically. The epidemiology of methicillin-resistant  Infectious disease surveillance is an eye to the public health. The fundamental epidemiology of infectious diseases can be understood using the surveillance data, which is critical to evaluate and plan the interventions needed to manage the target diseases. Antimicrobial resistance (AMR) is now recognized as a global public health issue. One of the five strategic objectives of the Global Action Plan of AMR is “strengthen the knowledge and evidence base through surveillance and research” . Staphylococcus aureus (MRSA), multidrug-resistant Pseudomonas aeruginosa (MDRP), vancomycin-resistant enterococci (VRE), multidrug-resistant Acinetobacter sp. (MDRA), and carbapenem-resistant Enterobacterales (CRE) in Japan.Japan has two national surveillance systems implemented by the Ministry of Health, Labour and Welfare (MHLW) to monitor the trend of AMR bacteria, which have been in effect for the last 20 years. This review describes the epidemiology of representative AMR bacteria as causative pathogens of healthcare-associated infections based on these surveillance data. It will focus on methicillin-resistant The current system of the National Epidemiological Surveillance of Infectious Diseases (NESID) has been operational since April 1999, when the “Law Concerning the Prevention of Infectious Diseases and Medical Care for Patients of Infections (Infectious Diseases Control Law)” was implemented. Since then, several amendments have been made to the Infectious Diseases Control Law, and 113 infectious diseases (as of August 2021) designated as “reportable infectious diseases” have been targeted under NESID. Those diseases are classified in the categories I to V based on the control measures and public health impact . The conhttps://www.niid.go.jp/niid/ja/idwr.html, accessed on 29 September 2021).Cases of notifiable and sentinel surveillance diseases are first reported to the health center in a prescribed form. The health centers report the accepted cases to local public health authorities, usually at the prefecture level, through the NESID web-based system. The surveillance data are finally reported at the national level and released as the Infectious Diseases Weekly Report (IDWR) on the website of the National Institute of Infectious Diseases, Japan (Streptococcus pneumoniae (PRSP)) are sentinel surveillance diseases reported by the sentinel hospitals on a monthly basis. At least one sentinel hospital exists in each medical region, and there are approximately 500 sentinel hospitals across Japan. The other four  are notifiable diseases, for which all cases are to be reported and can be regarded as population-based surveillance. PRSP is an important AMR bacteria; however, because PRSP infection is a community-acquired infection rather than a healthcare-associated infection, a description of its epidemiology is out of the scope of this review. Additionally, no case of VRSA has been reported officially in Japan, and thus, there is no corresponding data of it.As per surveillance definition, AMR is included for seven infectious diseases, all of which belong to category V . Of themJapan Nosocomial Infections Surveillance (JANIS) was launched in 2000 as a voluntary surveillance system focusing on infections in healthcare settings in Japan. JANIS is not based on the Infectious Diseases Control Law, but is funded by the MHLW and managed by the National Institute of Infectious Diseases. There are five divisions in the JANIS system with distinct surveillance objectives. Among them, the JANIS clinical laboratory division (JANIS-CL) is a surveillance system, which specifically focuses on AMR bacteria.https://janis.mhlw.go.jp, accessed on 29 September 2021). The details of the JANIS system have been described previously ,3. The uUnlike the typical infectious disease surveillance, JANIS-CL does not include case definition for reporting. However, it defines each AMR bacteria  based on species identification and AST results. Generally, these definitions are consistent with those defined by laboratory results per NESID case definitions. Conversely, the most important difference between NESID and JANIS-CL is that NESID cases include patients with symptomatic infections, whereas JANIS-CL includes both symptomatic and asymptomatic/colonized patients. Moreover, JANIS-CL division data cannot differentiate symptomatic and asymptomatic/colonized patients. For AMR bacteria, which are not targeted by NESID, the interpretation of AST results (susceptible or resistant) is mainly performed based on the Clinical and Laboratory Standards Institute (CLSI). The annual “Open Report” includes several types of aggregated data to assess the distribution of AMR bacteria. The prevalence of AMR bacteria in JANIS-CL is expressed as follows: the numerator denotes the number of patients with the specific AMR bacteria and the denominator denotes the number of patients whose clinical specimens for microbiological tests were submitted. The dissemination level of each AMR bacteria can be evaluated through assessing the proportion of hospitals reporting the particular AMR bacteria among all JANIS-CL participating hospitals. When this proportion is high, the corresponding AMR bacteria is considered endemic and disseminated widely. Conversely, when it is low, the dissemination of the corresponding AMR bacteria is considered limited. Finally, the antimicrobial susceptibility pattern based on bacterial species, usually called antibiograms, has also been depicted for major bacterial species. The number of JANIS participating hospitals has increased since 2007, and as of January 2021, more than 2000 hospitals are included in JANIS-CL. It accounts for almost a quarter of all hospitals . Additionally, focusing on larger hospitals with more than 500 beds, which usually include the tertiary care, general, or teaching hospitals, more than 80% of those hospitals are participating in this surveillance system. Therefore, JANIS-CL division data have good representation, especially of the tertiary care hospitals in Japan.S. aureus isolated from the clinical specimens were MRSA [MRSA is a representative AMR bacteria in Japan, and its emergence and rapid spread have made Japanese people realize the importance of infection control in healthcare settings, not only by the healthcare workers, but also by the general public. ere MRSA . MRSA isMRSA infection cases reported to NESID per sentinel are shown in In spite of these policies and developed human resources, the decrease in MRSA incidence slowed down in the late 2010s. A possible reason could be inferred from JANIS-CL data. Unlike NESID, which includes data only from sentinel hospitals , JANIS-CL receives data from small hospitals with less than 200 beds. In The MRSA epidemic started from large hospitals and rapidly spread to small hospitals by transferring patients , and it It is noteworthy that the reimbursement of 4000 Yen per patient per admission for implementing infection control practices is mainly targeted at the large hospitals. Conversely, of the 8300 hospitals in Japan, 5769 (69.5%) are small hospitals with less than 200 beds. Usually, the small hospitals are privately owned and provide both acute- and long-term care. Smaller reimbursement (1000 Yen per patient per admission) for implementing infection control practices in the small hospitals also exists with minimal requirements. Infection control in mixed or long-term care facilities with limited resource needs different strategies than in acute care hospitals, making it an urgent and important issue requiring intervention in Japan.P. aeruginosa resistant to carbapenems, fluoroquinolones, and amikacin  in the 2010s; however, it increased to 0.04% since 2019 . Of notepstS-null Enterococcus faecium) first reported in Australia is suggested [Since 2019, several VRE outbreaks with more than 100 cases per hospital have been reported, which is unusual in Japan. These VRE outbreaks started from a single hospital but spread rapidly within the entire medical region. Both NESID and JANIS-CL data reflected these outbreaks, and their rapid dissemination as the number of reported VRE cases recorded were the highest in 2020, and an increase in VRE prevalence was recorded in JANIS-CL . The reauggested ,22,23. IThe prevalence of MDRA in Japan still remains quite low. Cases of MDRA infection were initially (February 2011) designated as sentinel reporting diseases in NESID; however, because of the low prevalence, it was changed to notifiable diseases in September 2014. In 2015, 38 cases were reported and the number of cases continuously decreased as there were only 10 cases reported in 2020 . Among 1In the 2020 JANIS-CL annual report, the prevalence of MDRA was 0.003% , reflecting the low prevalence, and only 21 (1.0%) hospitals out of 2167 JANIS participating hospitals reported MDRA.Although the dissemination of MDRA in Japan is limited, it did play an important role in implementing the infection control policy. In 2010, an outbreak of MDRA with 53 cases including nine fatalities occurred . The hosThe rapid dissemination of NDM-type carbapenemase-producing Enterobacterales (CPE) reported in 2010 made CRE widely recognizable as an AMR pathogen of concern. This was also reported by the Japanese mass media together with the first report of NDM-type CPE isolation in Japan . CRE infKlebsiella aerogenes, while that among CPE is Enterobacter cloacae complex. Additionally, IMP-type is the dominant carbapenemase. The dominance of IMP-type carbapenemase is different from Europe, the United States, or other Asian countries where OXA-48-like, KPC- or NDM-type CPE are more dominant types of carbapenemase. In Japan, those types of CPEs are thought to be an imported-type of AMR. However, for NDM-type CPE, cases without international travel have been reported, which is suggestive of local transmission. The dominance of K. aerogenes may be due to the unique definition of CRE in Japan  , which dBoth NESID and JANIS-CL are AMR surveillance systems, which were optimized for Japan. Therefore, they were sustained for more than 20 years with verified data and high representativeness. However, the number of reported cases of AMR bacterial infections in NESID and prevalence of AMR bacteria calculated in JANIS-CL is hardly comparable with those of other countries. It is because the definitions of AMR bacteria are different  and different denominators, such as the number of specimens-submitting patients, as used in JANIS-CL.P. aeruginosa and Acinetobacter spp. was adapted instead of MDRP and MDRA, respectively, and E. faecium was considered as a representative bacterial species of VRE. For CRE, K. pneumoniae was used as a representative bacterial species, because it is the most prevalent bacterial species in European countries, although it is not the most dominant bacterial species in Japan. Those comparing charts were organized by the period of their epidemic or emergence/spread.Conversely, data for antibiograms in the JANIS-CL division, which indicate antimicrobial-resistant rate based on the bacteria species for several antimicrobials independently, are more comparable internationally. As described previously, it follows CLSI for SIR  interpretation. It seems that Japan has succeeded in controlling AMR bacteria causing healthcare-associated infections which emerged and spread after the 2000s, such as VRE and MDRA. This could be accomplished because of the improvement of infection control practices, and training and deployment of infection control professionals in the 2000s . HoweverThe COVID-19 epidemic, which started in early 2020, has induced drastic behavioral changes in people and caused a significant decrease in other infectious diseases like influenza. The behavioral changes may also include healthcare-seeking behavior. The number of hospitalized patients per day decreased by almost 7–8% since the COVID-19 epidemic. It can also be seen in JANIS data, as the number of patients submitting specimens decreased by 7.2% in 2020 compared to 2019. Furthermore, reports of COVID-19 outbreaks in healthcare settings may have strengthened infection prevention practices, especially standard and droplet infection precautions. Although more careful study is required, these may explain the slight decrease of MRSA, MDRP, and CRE cases in both NESID and JANIS in 2020. In terms of MDRA, the decrease in the reported cases in NESID may be largely attributed to the decrease in imported MDRA cases from overseas.Conversely, as described in VRE section, Japan is now facing the nationwide epidemic of VRE. This may indicate that, to contain VRE dissemination, more pathogen-focused infection control measures, including strengthening contact precaution, active surveillance to detect VRE colonized patients combined with adequate isolation and cohorting, are required. Additionally, even if regional VRE outbreaks were detected, it was difficult for health centers and hospitals to focus on VRE outbreak management during the COVID-19 epidemic. The careful assessment of AMR surveillance data during and after the COVID-19 epidemic is important for controlling AMR in the future.It has been more than three decades since the Japanese medical setting has been facing an MRSA epidemic as a cause of healthcare-associated infections and two decades since the surveillance systems to monitor AMR bacteria were launched. The accumulation of nationwide, representative surveillance data of AMR for the past 20 years in Japan has contributed to our knowledge of AMR epidemiology, which is applicable for evidence-based intervention. AMR bacteria are sometimes described collectively. However, specific AMR bacteria, such as MRSA, MDRP, and VRE, exhibit different epidemiology, and therefore, the focus point for intervention can be different. Therefore, healthcare professionals in this field need to understand the importance of surveillance and cooperate in surveillance data collection. In addition, it is a responsibility of the public health authorities to maintain a good surveillance system for AMR bacteria and share the data and findings with the healthcare professionals and academicians to improve evidence-based infection control measures.


The variations in the measured delays can be directly related to the different heights of the potential barriers that the outgoing electrons see in the vicinity of shape resonances. Our results indicate the possibility to investigate the spatial characteristics of the molecular potential by mapping attosecond photoionisation time delays in the recoil-frame.Photoionisation time delays carry structural and dynamical information on the target system, including electronic correlation effects in atoms and molecules and electron transport properties at interfaces. In molecules, the electrostatic potential experienced by an outgoing electron depends on the emission direction, which should thus lead to anisotropic time delays. To isolate this effect, information on the orientation of the molecule at the photoionisation instant is required. Here we show how attosecond time delays reflect the anisotropic molecular potential landscape in CF Photoionization time delays provide insights on the interaction of photon with the electrons. Here the authors explore the role of the molecular potential to the attosecond time delays in the photoionization of CF4 molecule. In general, the electronic potential presents a non-spherical shape, which plays a key role in the stereo-dynamics of atom-molecule collisions3. In atoms and molecules, this charge can be one of the electrons contained in the system, which must absorb enough energy from an external source to overcome the ionisation potential and to acquire the necessary kinetic energy to explore the potential landscape, while staying long enough in the molecular surroundings to sample its relevant features. This is ideally possible by using ultraviolet radiation, i.e. photon energies of a few tens eV. In a classical picture, an electron with 10 eV energy takes about 53 as to travel through the typical molecular extension of 1 Å. The extremely short timescale of this motion calls for the application of attosecond pulses, which can efficiently generate photoelectron wave packets and provide the necessary time resolution7.As explained in textbooks, the effect and the spatial gradient of a potential can be unveiled by monitoring the motion of a probe charge immersed in that potential8. Using this approach, the role of electronic correlation effects in the photoionisation of atoms has been investigated in real-time11. Attosecond time delays have also been reported in photoionisation in molecular systems, showing the relevance of nuclear motion in hydrogen12 and the role played by shape resonances in N2O13 and nitrogen15. Moreover, the role of the localisation of the initial wave function16 and of a functional molecular group17 has also been demonstrated.The dynamics of photoionising wave packets is usually investigated by means of pump-probe experiments, in which an isolated or a train of attosecond pulses in the extreme ultraviolet (XUV) range set the photoelectron wave packet free and a synchronised infra-red (IR) pulse probes the instant at which the electron enters the continuum18) and a measurement-induced contribution due to the action of the IR probe pulse on the photoelectron wave packet moving in the long-range Coulomb potential20. While in atoms the influence of the latter term can be usually quantified through simple formulas independent of the specific target20 and its angular dependence has been characterised21, in molecules the effect of the IR field on the measured time delays has not been characterised yet. In general, in the case of molecular systems, the contributions of the two terms cannot be disentangled22, which requires a more involved analysis.In atomic systems, the photoionisation time delays are usually decomposed into a term specific of the atomic potential  of ionic fragment(s) after the interaction with the XUV radiation, which defines the recoil frame. Symmetric molecules consisting of only a few atoms are ideal to test these effects in this frame. On the one hand, small molecules present a limited number of photoionisation and photofragmentation pathways, making feasible the identification of the electronic level of the outgoing photoelectron and, under suitable conditions, the determination of the molecular orientation during the interaction with the ionising radiation. On the other hand, the symmetry of the molecule gives the opportunity to identify specific privileged directions in the recoil frame and to characterise the effect of the molecular potential along them.4 molecules by means of photoelectron-photoion coincidence spectroscopy23. The advantage of this approach is the possibility to derive information on the molecular orientation at the instant of photoionisation by measuring in coincidence the momenta of the emitted electron and the fragment ions resulting from the ulterior dissociation of the molecular cation24. In this way, we have been able to unambiguously identify individual ionisation channels and obtain time-resolved recoil-frame photoelectron angular distributions (RFPADs) from which the variations of photoionisation delays with the electron emission direction have been extracted. The measured delays are in very good agreement with those obtained from calculated time-resolved RFPADs where all the transitions induced by the attosecond pulse train and the IR probe, in particular those induced in the continuum by the IR pulse, are taken into account in a time-dependent formalism. The agreement confirms the validity of our experimental approach and opens the route to orientation-specific exploration and understanding of molecular photoionisation delays.In this work we investigate the photoionisation dynamics induced by a train of attosecond XUV pulses on CF4, which is triply degenerate and belongs to the irreducible representation T1 of the point group Td  and α .We focus on photoelectrons emitted from the Highest-Occupied Molecular Orbital of CF25. This approach usually called RABBITT (Reconstruction of attosecond beating by two-photon transitions)8 allows one to combine temporal and spectral resolution, which turns out to be advantageous for identifying the different fragmentation channels or yields involved in the experiment. The setup used in the measurements is described in ref. 26.Photoelectrons and photoions emitted after the interaction with an attosecond pulse train generated in argon and krypton and a synchronised IR field were measured using a Reaction Microscope4 into five different cationic states  is energetically possible27. While the first three predominantly lead to the formation of CF28. Additional correlation between the kinetic energy release (KER) of the ions and the photoelectrons gives the possibility to isolate the contribution of the photoelectrons associated with the X2T1 state from those associated with the A2T2 and B2E states  results in fast dissociation and emission of CF29, giving access to the relative orientation of the polarisation direction of the external fields with respect to the polar angle β  at the instant of photoionisation using the recoil approximation30. The validity of the approximation is further confirmed by the good agreement between the measured photoelectron angular distributions (PADs) for this state and those calculated by assuming a fixed nuclei configuration (see below). Because the position of the fluorine atoms in the tripod is not determined in the experiment, the measured PADs are plotted as a function of the θ angle, i.e. with respect to the z-axis  or perpendicular (60∘ ≤ β ≤ 120∘) with respect to the light polarisation vector. The signal is therefore obtained by integrating over all possible photoelectron emission directions, but for the specific molecular orientations with respect to the light as indicated in the right side insets of Fig. θ in the recoil frame.The photoelectron spectra measured as a function of the delay between the XUV and IR pulses for specific molecular orientations with respect to the light polarisation vector of the collinear fields are presented in Fig. mentclass2pt{minimθ are reported in Fig. θ for the parallel and perpendicular cases were estimated from Fig. The attosecond time delays determined for different recoil frame emission angles 31 and with laser parameters that reproduce the experimental conditions (see details in the SI), are also shown in Fig. The theoretical results obtained by solving the TDSE within the static-exchange DFT method described inθ = 90∘, while they are relatively independent of θ in the perpendicular case , B (θ = 68∘) and C (θ = 165∘), respectively. Photoelectrons leaving the molecule along these three directions, experience significant differences in the molecular landscape, as shown in Fig. For the parallel case  and B and C (ΔτB−C = τB − τC), respectively. The experimental values (red squares) were determined integrating the photoelectrons emitted in the parallel configuration over the emission angles θ in the intervals θA = [0−29∘], θB = [60−75.5∘], and θC = [151−180∘] along the directions A, B, and C, respectively. The theoretical values (black circles) were obtained from the calculated RABBITT spectra in the same way. The delay difference ΔτA−C presents a minimum in the experimental as well as in the theoretical data around 34 eV photon energy, which approximately matches the maximum of the shape resonance for the direction C observed in Fig. τA−C becomes smaller.The anisotropic molecular potential influences the photoionisation dynamics, as demonstrated in Fig. 33 obtained from the one-photon (XUV) dipole matrix elements and integrated over the corresponding angle intervals is also reported (blue triangles) and indicates a minimum in the same energy region. The deviations between the difference of the stereo Wigner time delays and the corresponding time delays obtained for the two-colour simulations, support the conclusion that the delay in photoionisation of molecules cannot be decomposed, in general, into the sum of a contribution due to the Wigner delay and one associated with the continuum-continuum delay22. The latter, indeed, should cancel out when subtracting the delay estimated along the directions A and C. In any case, the overall evolution of the delay is qualitatively similar suggesting the relevance of the differences in the position of the shape resonances along the directions A and C in the photoionization process. Differently from the previous case, the experimental data for the delay difference ΔτB−C (red square) do not show any significant variation of the delay as a function of the photon energy, as expected. The theoretical values (black circle) are in good agreement with the experimental data, indicating only a moderate linear increase of the delay. The absence of any remarkable variation of the delay as a function of the sidebands for these two directions can be attributed to the similar heights of the trapping potentials, as shown in Fig. The experimental points are in good agreement with the theoretical curve (except for the point corresponding to sideband 20). The stereo Wigner time delayWe have demonstrated that the anisotropic molecular landscape affects the stereo-photoemission time delays. In particular, different heights of the trapping potentials introduce different delays in the emission of the photoelectron wave packet into the continuum. Our results indicate the importance of coincidence spectroscopy to disentangle the information on the photoemission process from different electronic states and for different molecular orientations. Extension of this approach would be beneficial also for larger and more complex molecular systems.The experiment was performed using a 10 kHz laser Ti:sapphire system providing 30-fs laser pulses centred at 800 nm with a pulse energy of 1 mJ. The input pulse first passes through a 1-mm-thick glass plate with a 3-mm-diameter hole at the centre splitting the beam into two parts , which are spatially separated and temporally delayed. Then, the input beam is focused by 25-cm-focal-length parabolic mirror into a high-harmonic gas cell filled with argon (Ar) or krypton (Kr) to generate XUV photons (20–46 eV) consisting of odd multiples of the input pulse frequency.26. For the XUV-only measurements, an aluminium filter is introduced into the beam propagation direction after the high-order harmonic generation cell in order to block the co-propagating IR pulse. Then, the XUV and probe pulses are refocused by a toroidal mirror into the interaction region inside a 3D momentum imaging spectrometer leading to dissociative photoionisation of CF4 at an ion count rate of about 0.1 per XUV pulse. The resulting ions and electrons are guided using a homogeneous electric field (∣E∣ ~ 313 V/m) and a weak magnetic field (∣B∣ ~ 9.4 G) towards time- and position-sensitive detectors located at the opposite ends of the spectrometer.In our work, the XUV photons were generated by the annular beam. By using an iris before the gas cell and blocking the annular beam, we ensure that the central beam, after focusing it into the gas cell, is not contributing to the XUV emission. After the gas cell, another 1-mm-thick glass plate with a 1-mm-diameter hole, centred with respect to the first plate, is placed to synchronise the XUV and IR probe pulses. The IR probe pulse is part of the input beam which is going through the hole of the first plate and glass of the second one. The delay between XUV and IR probe pulse is varied precisely by tilting the second, drilled plate31. In this method, the time-dependent wave function is expanded in a basis set of N-electron stationary wave functions, built from antisymmetrized products of an (N − 1)-electron wave function for the bound electrons and a one-electron continuum wave function for the electron ejected into the continuum. The (N − 1)electron wave functions are built from B-spline representations of the lowest Kohn-Sham (KS) orbitals resulting from the diagonalization of the KS hamiltonian of density functional theory, excluding the orbital from which the electron is ejected to the continuum, and the one-electron continuum wave functions are obtained from an inverse iterative procedure in the former KS orbital basis for each photoelectron energy. All dipole matrix elements describing the transitions between all bound and continuum states as well as between continuum states have been included in the solution of the TDSE, which is essential to describe the bound-continuum and continuum–continuum transitions leading to the RABBITT spectra. We have restricted all simulations to the equilibrium geometry (fixed-nuclei approximation). More details can be found in the In brief, theoretical RABBITT spectra have been obtained by solving the time-dependent Schrödinger equation (TDSE) within the static-exchange approximationSuppelementary Information


By releasing 20% of the loaded telmisartan in 30 days, these hybrid biodegradable/stent telmisartan-loaded nanofibers increased the migration of endothelial progenitor cells in vitro, promoted endothelialization, and reduced intimal hyperplasia. As such, this work provides insights into the use of PLGA nanofibers for treating patients with an increased risk of stent restenosis.Stent implantation impairs local endothelial function and may be associated with subsequent adverse cardiovascular events. Telmisartan, an angiotensin II receptor blocker that has unique peroxisome proliferator-activated-receptor-gamma-mediated effects on cardiovascular disease, has been shown to enhance endothelial function and limit neointimal hyperplasia. This study utilized hybrid biodegradable/stent nanofibers to facilitate sustained and local delivery of telmisartan to injured arterial vessels. Telmisartan and poly( Catheter-based interventional strategies represent the gold standard for revascularizing artery stenosis. However, balloon angioplasty or limus-eluting stent implantation result in damage to endothelial cells ,2. The fTelmisartan, an angiotensin II receptor blocker that is routinely used in clinical practice, can improve the function of endothelial cells, reduce responses to oxidative stress, and reduce superoxide-caused damage ,11. The d,l)-lactide-co-glycolide (PLGA) nanofibers/stent for the local and prolonged delivery of telmisartan into a rabbit denuded artery. Electrospinning is a cost-effective and efficient process that produces continuous nanoscale fibers with diameters in the sub-micrometer to nanometer scale [This work develops biodegradable, electrospun hybrid poly was dissolved into 1 mL of HFIP for the nanofiber electrospinning [PLGA  has a lactide:glycolide ratio of 3:1 and a molecular weight of 76,000–115,000 Da. Telmisartan was purchased from Sigma-Aldrich . Following our previous work ,22, hexaspinning . A syrinnanofibers and ρpolymer denote the densities of the nanofibers and the polymer, respectively.The porosity of the nanofibers was calculated using the following equation.n = 3) utilizing ImageJ .Electrospun biodegradable nanofibers were first coated with gold and then characterized by an SEM. Size distribution of spun nanofibers was assayed from one hundred randomly chosen fibers (n = 4) on each scaffold. The elongation and tensile strength at breakage were obtained as follows.The mechanical properties of nanofibrous scaffolds were measured using a Lloyd tensiometer  with a 0.1 kN load cell using the ASTM D638 standard. A strip with an area of 50 mm by 10 mm from the scaffolds was used and gripped between two clamps 30 mm apart. The material was moved by the top clamp at 60 mm/min through a distance of 10 cm before the clamp was returned to its starting point. The elongation of the samples and the force on the scaffolds upon breaking of the scaffolds were recorded. The test was conducted four times (n = 4).Using a water contact angle analyzer , the wetting angles of the scaffolds were obtained. An area of 10 mm by 10 mm were prepared. Distilled water with a volume of 0.1 mL was lightly dropped onto the surfaces of the testing area and examined with a video monitor  is calculated as follows.The in vitro release properties of telmisartan from the electrospun nanofibers were obtained using an elution method. The samples loaded with telmisartan were put in test tubes with 1 mL of PBS. Following a 24 h incubation at 37 °C, the eluent was analyzed. The PBS was replaced and the test tubes were incubated for another 24 h before the eluent was collected and analyzed. This process was repeated over 30 days.8, 3.9 cm × 150 mm HPLC column (Waters) was used to separate out for the separation of telmisartan. The mobile phase used for telmisartan was (0.05 M KH2PO4 + 1 mL phosphoric acid): acetonitrile 40:60 (v/v). The absorbency was monitored at a wavelength of 271 nm and the flow rate was 1.0 mL/min.Using a high-performance liquid chromatography (HPLC) assay, the telmisartan concentrations in the eluents were obtained. The HPLC analyses were performed using a Hitachi L-2200 Multisolvent Delivery System . A SYMMETRY C2 to allow cell attachment. After a 24-h incubation, the medium was replaced with 600 μL Dulbecco’s modified Eagle medium (DMEM)  that was supplemented with 2% (v/v) FBS , 100 U/mL penicillin, and 100 μg/mL streptomycin  and cells were cultured for another 24 h. On the following day, 6 × 104 EPCs were seeded into the upper chamber of the transwell inserts. The culture was then maintained for two hours at 37 °C in a humidified atmosphere that contained 5% CO2. At the end of the process, the number of EPCs that had migrated to the other side of the transwell inserts was determined by hematoxylin staining. The EPCs that were cultured in transwells where the lower chambers contained cell-free DMEM served as controls.The effect of the nanofiber-released telmisartan on endothelial progenitor cell (EPC) migration was then quantified ,27,28,29Adult male New Zealand white rabbits with a mean mass of 3.22 ± 0.24 kg were used throughout the in vivo portions of this study. They were all housed in separate cages in a light- and temperature-controlled room and had free access to sterilized drinking water with standard rabbit chow ad libitum. In accordance with the regulations of the National Institute of Health of Taiwan, under the supervision of a licensed veterinarian, all animal procedures were institutionally approved and all of the studied animals were cared for (Chang Gung University CGU14-045).Using a muscular injection of Zoletil 50  and xylazine (9.3 mg/kg), and oxygen (2 L/min) through a face mask, rabbits were anesthetized and sedated. Group A consisted of 12 rabbits with telmisartan-loaded hybrid stents; group B consisted of 12 rabbits with hybrid stents which did not contain telmisartan. The rabbits were treated with an endothelial denudation of the descending abdominal aorta using a 3.5 × 20 mm Maverick balloon  to create an arterial balloon injury. After stent deployment, angiography was used to determine post-operative vessel patency. To satisfy institutional regulations and ethical concerns, aspirin (40 mg/d) was administered orally 24 h before catheterization and then continuously throughout the study. A single dose of intra-arterial heparin (150 IU/kg) was administered during the procedure.Stented vessels were gathered at one month following deployment for microscopic observation and histological examination.Intact stented vessels were rinsed in 0.1 mmol/L sodium phosphate buffer (pH 7.2 ± 0.1) and subsequently post-fixed in 1% osmium tetroxide for approximately 30 min. They were then dehydrated in a graded series of ethanol solutions. All the tissue samples were mounted, sputter-coated with gold, and observed under an SEM .The degree of endothelialization of the stented area were estimated using low-power photographs of the lumen surface at a magnification of 35×. Increased magnification (200×) was then used to directly visualize the endothelial cells. The extent of the endothelial surface coverage on the stent struts was characterized by ImageJ  .p value < 0.05. Data were analyzed using SPSS software .All data are presented as mean ± standard deviation. One-way ANOVA was used to compare data to identify statistically significant relationships. Within ANOVA, the post hoc Bonferroni procedure for multiple comparisons was used to identify significant differences between pairs. Differences were regarded as statistically significant at Hybrid biodegradable telmisartan-loaded nanofibers were developed using PLGA 756 and electrospinning. PLGA is a thoroughly-characterized, biodegradable, and biocompatible copolymer with medical applications that has been widely used in drug delivery systems ,31. Nanop < 0.001, p = 0.041, p = 0.744) after a half-hour, while that of the telmisartan/PLGA 756 nanofibers peaked at 86.7 ± 18.4% at one hour . Thereafter, the water content in both groups declined with time . Additionally, the porosity of the nanofibrous membranes in the telmisartan group was higher (76.4 ± 5.0%) than that in the plain PLGA756 group (58.4 ± 6.6%) (p = 0.005)  h. High pIn aqueous environments, through bulk or heterogeneous erosion, PLGA is degraded by hydrolysis of its ester linkages. A compound’s release rate depends on the properties of the polymer and drugs in the physiological environment ,45. In gp = 0.010) (p < 0.001). As such, the two nanofibrous membranes have adequate properties for stent expansion stress tolerance.Optimal stent implantation requires high inflation pressures and high-pressure angioplasty, defined as dilation at a pressure of 18 atm  (approxi= 0.010) b. Additip < 0.001).Endothelial progenitor cells (EPCs) form a circulating pool of cells that can create a cellular patch at sites of endothelial injury, contributing directly to the homeostasis and repair of the endothelial layer. Virtually all risk factors for atherosclerosis have been associated with a decrease in either the abundance, the dysfunction, or both, of circulating EPCs ,49. Telmp = 0.002) (p = 0.001) (= 0.002) g. After = 0.001) h.p < 0.001). Heme oxygenase-1 (HO-1) plays a critical role in the prevention of vascular inflammation and may protect against atherosclerosis by preserving vascular cell function and survival [p < 0.001). Therefore, telmisartan may induce an upregulation of HO-1 which blocks the inflammatory response in vascular cells and potently inhibits vascular smooth muscle cell proliferation.The relative ratio of HO-1 around the stenting area at four weeks post-surgery is shown in survival . Endothesurvival . InteresElectrospun hybrid biodegradable/stent telmisartan-loaded nanofibers significantly improved various metrics pertinent to vessel revascularization. In vitro and in vivo analyses of hybrid stent/PLGA 756 nanofibers with telmisartan loading indicate that they can controllably release disease-relevant therapeutics, enhance the migration of EPCs, provide complete endothelial coverage and recovery, and reduce intimal hyperplasia. Taken together, these findings suggest that hybrid stents/telmisartan-loaded nanofibers have widespread potential applications in cardiovascular drug delivery. Specifically, the use of hybrid nanofibers with the capacity for sustained and local drug delivery may prove particularly useful in the treatment of patients with an increased risk of arterial restenosis.