Stretchable optical fiber sensors (SOFSs), which are promising and ultra-sensitive next-generation sensors, have achieved prominent success in applications including health monitoring, robotics, and biological–electronic interfaces. Here, we report an ultra-sensitive multi-functional optical micro/nanofiber embedded with a flexible polydimethylsiloxane (PDMS) membrane, which is compatible with wearable optical sensors. Based on the effect of a strong evanescent field, the as-fabricated SOFS is highly sensitive to strain, achieving high sensitivity with a peak gauge factor of 450. In addition, considering the large negative thermo-optic coefficient of PDMS, temperature measurements in the range of 30 to 60 °C were realized, resulting in a 0.02 dBm/°C response. In addition, wide-range detection of humidity was demonstrated by a peak sensitivity of 0.5 dB/% RH, with less than 10% variation at each humidity stage. The robust sensing performance, together with the flexibility, enables the real-time monitoring of pulse, body temperature, and respiration. This as-fabricated SOFS provides significant potential for the practical application of wearable healthcare sensors. At present, the research and application of flexible wearable electronics are reflected in various aspects of human life ,2,3,4, sIn addition to traditional electrical sensing, combining optical sensing with soft and stretchable packaging may provide an alternative means of developing cheap but reliable wearable sensors ,23. WithTo address this issue, we propose a method to fabricate multi-functional stretchable fiber sensors by using a PDMS membrane embedded with MNFs. Based on the transition from guided modes to radiation modes of the waveguiding MNFs upon external stimuli, the as-fabricated MNF sensors are demonstrated to have outstanding sensitivity and repeatability when used in strain and temperature sensing. Moreover, by taking advantage of the water-absorbing ability of tin oxide, a wide detection range and highly sensitive humidity sensing are realized.In order to fabricate the MNFs, we used an electricity-heated mechanical stretching technique to draw a standard silica optical fiber into a biconical tapered fiber. In this method, a 3 cm-long section of the standard fiber was stripped of its cladding layer and fixed onto the MNF translation platform. When the fiber was heated to an optimal temperature, it was drawn into the horizontal plane until the diameter was reduced to the desired value. The entire MNF fabrication process was controlled by a computer program. To obtain uniformity among MNFs with different diameters, the temperature distribution in the drawing region and the speed of the stepper motors could be adjusted.To fabricate an SOFS, we developed a sandwich-like structure to encapsulate the as-fabricated MNFs, which involved a three-step process: (1) 25 um of PDMS was prepared on a thin polyethylene (PET) flake to support the MNFs, which was firmly held in position using van der Waals forces and electrostatic interactions between the MNFs and the PDMS membrane; (2) 1 mL of degassed PDMS  was slowly poured onto the PDMS/PET substrate, waiting for several seconds to ensure the substrate was covered with the fluid PDMS and that the MNFs were enclosed in the liquid; and (3) another thin PDMS/PET flake was placed on the substrate, followed by curing at 80 °C for 30 min, forming a 500 um-thick PDMS thin film. As for humidity sensing, a humidity-sensitive layer (tin oxide) was deposited using the magnetron sputtering method on the as-fabricated MNFs; the thickness of the tin oxide layer was about 20 nm for the sake of optimal sensing performance. According to the requirement of various sensing applications, the as-fabricated SOFS was peeled from the PET sheet.Computer-controlled stepper motor translation platforms, which precisely control different step movements (including single step movement and periodic step movement), were prepared to measure the strain sensing performance of the SOFS. Before strain sensing, the as-fabricated SOFS was fastened onto the translation platforms with standard clamps and coupled with a 1550 nm wavelength fiber laser at one end. The optical output was detected by a spectrometer at the other end. The sensing process can be divided into two main parts: the first is a micro-strain measurement which contains several micron dimension step movements, and the other is an endurance test with hundreds of cycles of movements. Finally, in order to demonstrate the capability for health monitoring, the detection of cardiopalmus was conducted.A constant temperature heating facility was used to test the temperature sensing performance of the SOFS, which could provide a stable temperature environment (ranging from 30 to 60 °C). During the temperature sensing process, the as-fabricated SOFS was positioned within the facility at all times and was always in a closed environment. Moreover, in order to demonstrate the capability of the SOFS for health monitoring, the detection of body temperature was conducted. A humidity generator and a detector were prepared to support the measurement of the SOFS’s humidity sensing capability, which provided a continuous standard humidity atmosphere (ranging from 10 to 90% RH). Furthermore, for the duration of the humidity sensing process, the as-fabricated SOFS was placed in an airtight chamber. In order to demonstrate the capability for health monitoring, the detection of respiration was conducted.An optical fiber laser  and an optical fiber spectrometer  were used as a light source and a detector, respectively. A humidity generator  and a humidity detector  were used to generate standard humidity and measure the unknown environmental humidity, respectively. When a single-mode optical fiber  was coupled with the fiber laser, the input optical power of the MNF was about 16 mW, with a stability of less than 0.05%. The stepper motors of the optical fiber drawing platform  and the translation stage  were used to investigate the strain response of the sensor. A home-made system consisting of multiple channels was used for monitoring the response of the optical sensor to humidity, maintaining a relatively long recording period. A constant temperature heating facility  with a temperature resolution of 0.1 °C was provided to maintain a stable temperature in the range of 30 to 60 °C.Although the conventional PDMS embedding method has been widely employed ,33, demoExperimentally, highly uniform MNFs are fabricated through the tapered drawing of silica glass optical fibers, which allows for a guiding light at the micrometer scale. However, the as-fabricated MNFs, with a large proportion of evanescent fields exposed to the surroundings ,35, are −4 per °C), and thus the guided modes of the MNFs are sensitive to the surrounding variation of RI owing to the large fractional evanescent fields outside the MNF, ensuring the SOFS’s responsiveness to the change in temperature. Moreover, a wider expansion of aspects of sensing media could be compatible with the as-fabricated SOFS. Tin oxide, as a humidity-sensitive material, has already been used in humidity sensing [Bending a flexible SOFS embedded with PDMS can lead to a bending-dependent transmission, which includes large-scale winding and tiny vibrations, enabling strain sensing. The RI of PDMS is a function of the temperature due to its large negative thermo-optic coefficient  as a function of strain leads to a gauge factor (GF), where the gauge factor is defined as GF = (∆I/I0)/strain. Meanwhile, the detectivity of the SOFS was shown to be highly sensitive, with a gauge factor of 118 and higher compared to conductive core–shell aramid nanofibrils (GF:18.8) [To investigate strain sensing, firstly, the SOFS embedded with a 1 um-diameter MNF was fastened to a computer-controlled stepper motor translation platform for bending sensing. By taking advantage of the high-precision moving stage, we designed each step to correspond to a replacement of 1 um, 2 um, and 5 um with 10 times bend and 10 times recover. As shown in GF:18.8) , and silGF:18.8) . ConsideGF:18.8) . To demoThe long-term reversibility and stability of the SOFS were further investigated by automatically bending and recovering the sensor over 250 cycles using a computer-controlled moving platform. After the cycles, the sensor maintained a steady baseline intensity with a slight decrease of 5% d, which I/∆T, where ∆I is the increase in the output intensity, and ∆T is the change in the temperature, the sensor achieved a sensitivity as high as 0.02 dBm per °C, which is similar to that of wearable temperature sensors based on metal oxides [In addition to strain sensing, the refractive index characteristics of PDMS are promising for temperature sensing; it has a large negative thermo-optic coefficient, while the silica MNF shows a negligible change compared with that of PDMS. The SOFS with a 1 um-diameter MNF embedded in a 500 um-thick PDMS film was placed on a computer-controlled constant temperature heating facility, which provided a stable temperature in the range of 30 to 60 °C. The transmission spectra of the SOFS at different temperatures were recorded using a spectrometer a. The prl oxides . The decBesides the excellent stretchability and temperature response, an ultra-sensitive optical sensor with a wide range of humidity detection was obtained according to the Materials and Methods section. Interestingly, when we started to study the effect of different diameters on the humidity response, SOFSs with diameters that were too small or too large under the same thickness of the sensitive layer were demonstrated to be unresponsive. The phenomena of guided light scattering and optical absorption  close to the most sensitive area in order to ensure wet air penetrated into the humidity-sensitive material. Tin oxide, a promising humidity-sensitive material, has not only been widely used in the field of humidity sensing but also popularized in the field of opto-electronics for decades [I/∆H, where ∆I is the increase in the output intensity, and ∆H is the change in the relative humidity, the as-fabricated sensor exhibits a peak sensitivity of 0.5 dB/% RH, which is far better than a humidity sensor based on a hetero-core optical fiber [Compared to the above sensors, the pattern of encapsulation was discriminating to some extent, which included a spindly silt , the humidity response curve presents quite a sharp peak and an excellent reverting capacity. In addition, the as-fabricated SOFS possesses a better response speed (of a few seconds) compared with its electronic counterpart  under a wide humidity range.To exclude the disturbance of continuous humidity measurement and demonstrate the effect of the SOFS under all humidity conditions, the humidity stages from 30% to 90% RH were conducted individually. As shown in In summary, we have demonstrated an ultra-sensitive multi-functional optical micro/nanofiber embedded with a flexible encapsulation of PDMS, which possesses great potential in the field of wearable optical sensors. By taking advantage of the character of its strong evanescent field, the as-fabricated MNF embedded with a PDMS membrane is highly sensitive to weak bends, enabling a robust durability within hundreds of measurements and high sensitivity with a peak gauge factor of 450. At the same time, taking the large negative thermo-optic coefficient of PDMS into account, a temperature response in the range of 30 to 60 °C was realized, resulting in a resolution of 0.02 dBm/°C. In addition, a humidity measurement within a broad detection range from 30% to 90% RH was demonstrated, with a sensitivity of 0.5 dB/% RH, and less than 10% variation at each humidity stage. Moreover, the as-fabricated highly sensitive sensing performance with flexibility enables the real-time monitoring of pulse, body temperature, and respiration. The above results may pave the way towards future wearable optical sensors for health monitoring including real-time monitoring of pulse, respiration, and other aspects of life signs.


The graphene and TiO2 hybrid nanoparticles are dip-coated on a biodegradable thermoplastic poly(lactic acid) (PLA) substrate. The thermoplastic PLA is approved by the Food and Drug Administration (FDA), and is widely utilized in medical devices. The graphene/TiO2 coating is substantiated to transform the hydrophilic PLA film into superhydrophobic biomaterials that can help to reduce hazardous medical-device complications. The surface wettability of the graphene/TiO2 nanoparticle-coated PLA surface was evaluated by measuring the apparent water contact angle. The surface chemical composition and surface morphology were analyzed via Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The graphene/TiO2-coated PLA film achieved superhydrophobic properties by demonstrating a water contact angle greater than 150°. The water contact angle of the graphene/TiO2 coating increased along with the concentration of the nanoparticles and the ratio of TiO2 to graphene. Moreover, the graphene/TiO2 coating exhibited excellent durability, whereby the contact angle of the coated surface remained unchanged after water immersion for 24 h. The duration of the effectiveness of the superhydrophobic coating suggests its suitability for medical devices, for which a short duration of administration is involved. This study reports an easy-to-replicate and cost-effective method for fabricating superhydrophobic graphene/TiO2-coated surfaces, which additionally substantiates a potential solution for the manufacturing of biomaterials in the future.Materials with superhydrophobic surfaces have received vast attention in various industries due to their valuable properties, such as their self-cleaning and antifouling effects. These promising superhydrophobic properties are taken into high priority, particularly for medical devices and applications. The development of an ideal superhydrophobic surface is a challenging task and is constantly progressing. Various strategies have been introduced; however, a minority of them are cost-effective. This work presents a facile fabrication of the superhydrophobic surface by using graphene and titanium dioxide (TiO Superhydrophobic biomaterials have been commonly employed in various applications due to their non-wettability. The superhydrophobic effect of these materials is generally indicated by a high contact angle >150°) 2]. The f2) nanoparticles onto a poly(lactic acid) (PLA) substrate. This method is substantiated to be employed in the large-scale production of superhydrophobic biomaterials, particularly targeting medical devices.Developments of functional superhydrophobic surfaces are mostly inspired by nature, for example, by the hierarchical structure of the lotus leaf, which provides the infamous lotus leaf effect, as well as the water-repellent surfaces of insects ,12,13. V2 are introduced as the coating materials due to their tendency to improve hydrophobicity, and their high corrosion resistance and low toxicity [2 nanocomposite materials exhibit enhanced antibacterial activities by suppressing the growth of both gram-negative and gram-positive bacteria via photocatalytic activity and suppressing the generation of reactive oxygen species [Graphene and TiOtoxicity ,19,20,21 species ,24,25,26 species . The Foo species . Previou species ,30.2 coating that was fabricated in this work aims to target applications, particularly medical devices, that require self-cleaning properties. The rough topography of the graphene/TiO2 surface provides protrusions that trap an adequate amount of air layers within the surface structure and promote resistance to bacterial adhesion. This mechanism induces the inhibition of biofilm formation [2 have shown superior bactericidal effects and antifouling abilities [2 coating on PLA film via the dip-coating method. Herein, we propose that the artificial superhydrophobic surface fabricated via a simple step is appropriate for imparting into medical devices, for which self-cleaning and antimicrobial properties are desirable.The superhydrophobic graphene/TiOormation ,32. In abilities ,35,36,37bilities ,39. The bilities . The pre2/g) and titanium dioxide  were obtained from Sigma–Aldrich, MO, USA. Polydimethylsiloxane (PDMS) prepolymers and curing agents were purchased from the Dow Corning Corporation, Lansing, MI, USA. Poly(lactic acid) (3052 D) was purchased from Natureworks LLC, Saint Paul, MN, USA. Chemicals, including n-hexane  and chloroform (CHCl3), were obtained from Merck, Darmstadt, Germany, and were used as received.Graphene nanoplatelets  (PLA) film was prepared using PLA grade 3052 D. PLA pellets of 5 weight % were used to produce a transparent and resilient PLA film. The PLA pellets were dissolved in 30 mL of chloroform by stirring for 1 h at 50 °C. The clear PLA solution was cast onto a glass petri dish and dried at room temperature for 24 h.2 coating solution was prepared with different concentrations of nanoparticles  in 30 mL of hexane. Each concentration of the coating solution contained graphene nanoplatelets and TiO2 nanoparticles with ratios of 10:0, 9:1, 7:3, 5:5, 3:7, 1:9, and 0:10. Prior to mixing, 0.3 g of PDMS (10:1 ratio of base to curing agent) was added into the solution. The suspension was then stirred for 10 min, followed by ultrasonication for 15 min. Poly(lactic acid) film (2 cm × 4.5 cm) was dip-coated with the graphene/TiO2 solution, at a withdrawal speed of 2 mm/s, and dried at ambient temperature. The dip-coating process was repeated 3 times. The final product was cured in the oven at 50 °C for 1 h.The graphene/TiOThe static water contact angles on the surfaces were measured by using a goniometer . A droplet of distilled water with a volume of 10 µL was applied onto the surface of the films. The contact angle of the water droplet was measured immediately after droplet stability. The sessile water contact angles on both sides of the droplet were measured and the average value was recorded. The test was performed in triplicate.−1 with a resolution of 4 cm−1, and analyzed with LabSolutions IR Software (version 2.2). The surface morphology was assessed by using the scanning electron microscope .The surface chemical compositions were determined by using Fourier-transform infrared spectroscopy . The FTIR spectra were collected over a spectrum range of 400–4000 cm2-coated film to water exposure over a prolonged time. The graphene/TiO2-coated film was immersed in distilled water at room temperature for 24 h. The contact angle measurements were performed immediately after the samples were dried.A water immersion test was used to evaluate the resistance of the graphene/TiO2 coating on PLA film was successfully fabricated via dip coating. The graphene/TiO2 nanocomposites coating achieved superhydrophobic properties, as shown in 2 is illustrated via the high apparent water contact angle. The hydrophilic PLA film, with a contact angle of 77 ± 5°, transformed into a superhydrophobic surface (contact angle of >150°) upon the application of the graphene/titanium dioxide coating.The superhydrophobic graphene/TiO2 nanoparticles concomitantly improves the non-wettability of the surface. This result is in concordance with the statement mentioning that superhydrophobic properties are influenced by surface roughness and the chemical composition of the surface. In addition to the low surface energy, the graphene and TiO2 nanoparticles provide the hierarchical structure to the surface of PLA film and thus endow the modified surface with low wettability. The surface roughness, attributed to the micro/nano structures on the graphene/TiO2 surface, entrapped a sufficient air layer on the surface. Hence, high water contact angles are observed, due to the reduction of the contact point between the water droplet and the coated surface.This phenomenon suggests that the enhancement of the surface roughness by the graphene and TiO2 coating on the surface increased from 0.1% to 0.5%. Thereafter, the contact angle of the graphene/TiO2-coated PLA surface slightly decreased when the concentration rose to 0.7%, followed by a surge at 0.9%. In other words, the graphene/TiO2 coating alters the surface structure of the PLA film and transforms the surface-wetting properties from hydrophilic to hydrophobic, and later to superhydrophobic, as the concentration of graphene/TiO2 nanocomposites increases  was obtained when the ratio of graphene to TiO2 was equal. In concordance with previous studies [2 provided surface roughness to the coated surface. The addition of graphene further enhanced the textured surface by establishing the hierarchical nanostructure on the superhydrophobic surface. Therefore, both graphene and TiO2 exert a synergistic effect to produce a surface with superhydrophobic properties.It is noteworthy that, for the 0.5% hobicity . Howeverct angle . The sur studies ,25, the 2 solution increased, the contact angle of the graphene/titanium dioxide-coated PLA surface increased  − 1, where sf indicates the total area of the solid–liquid interface and θc represents the contact angle of the coated PLA surface, whereas θ represents the contact angle of the smooth PLA surface. By substituting 77°, which is the contact angle of the smooth unmodified PLA film, and the contact angles of the respective coated surface into the equation, the result for sf can be determined (sf value for the superhydrophobic surface is less than 0.1, which is in agreement with the previous studies reported by Karapanagiotis et al. and Basu et al. [2 allowed for a sufficient amount of air entrapment on the surface, thereby lowering the wettability and endowing the graphene/TiO2-coated surface with superhydrophobic properties.The hierarchically structured surface attributed to the graphene and TiOequation , cos θc termined . The resu et al. ,43 The s2 coating are characterized via FTIR, as illustrated in −1 to 3600 cm−1 represent the O−H stretching. The peaks around 1000 to 1200 cm−1 represent the C–O−C stretching of PLA. The peaks of ester are depicted at 1083 cm−1, belonging to the C–O−O stretching vibration, and circa 3000 cm−1, which belongs to the CH3 stretching band. Moreover, the asymmetric and symmetric C−H deformations in CH3 bending are depicted by the peaks at 1454 cm−1 and 1365 cm−1. The carbonyl C=O stretching is depicted as the peak at 1751 cm−1. The bands between 1630 cm−1 to 1800 cm−1 represent the C=C bond [2 produces an intensified peak at 1454 cm−1. The Ti−O stretching band can also be observed at 890 cm−1 and 750 cm−1 [−1 and 1253 cm−1, attributed to the in-plane and out-of-plane bending of the Si−CH3 [−1 and 3000 cm−1 represent C−H stretching, while the peaks between 1000 to 1400 cm−1 represent C−O stretching. The peaks around 1000 cm−1 to 1130 cm−1, which represent the Si–O–Si bands of PDMS, is suggested to be overlapped with the C−O-stretching bands.The chemical functional groups of the graphene/TiOC=C bond ,45,46,47750 cm−1  The peak2-coated film are illustrated in 2-coated surface shows a homogenous distribution of graphene and TiO2 nanoparticles. The uniform coverage of graphene and TiO2 nanoparticles provides a roughness to the surface, which is accountable for the low surface wettability. The graphene nanoplatelets are irregular in shape, whereas the TiO2 nanoparticles appear to have a nanoscopic particle size of less than 50 µm. The hierarchical structure on the coated surface is attributed to the graphene/TiO2 nanoparticles acting as protrusions. The interspacing between the protrusions allows for an adequate amount of air pockets to be entrapped on the surface. As depicted in the 3D images in 2 coating provides protrusions with different heights and interspacing, which help to minimize the contact point of liquid on the surface. This result correlates with the aforementioned porosity factor in 2 nanoparticles. The graphene/TiO2 hybrid nanocomposite confers a higher degree of surface roughness, therefore manifesting a greater reduction in the total surface area of the solid–liquid interface. The mixture of graphene and TiO2 nanoparticles offers an optimal surface roughness, which allows for a sufficient amount of air-layer entrapment, thus furnishing the coated surface with superhydrophobic properties. We suggest that the Cassie–Baxter regime be used for this scenario. The minimal solid–liquid interface, consequently, results in the high-water contact angle, and hence, the superhydrophobic properties.The surface morphologies of the non-coated PLA film and the graphene/TiO2-coated films are evaluated via the water immersion method, according to ASTM D870 [2-coated film (2-coated film decreased by approximately 2° after being immersed in water for 5 h. An additional period of water immersion, up to 24 h, further decreased the contact angles by approximately 5°. Nonetheless, the contact angles of the three different ratios of the graphene/TiO2-coated films remained above 150° at the end of the water immersion test. This indicates that the immersion of graphene/TiO2-coated film in water for a prolonged period does not result in a significant degradation of the coating. In the practical setting, it is not uncommon for medical devices to be in direct contact with body fluids for less than 8 h. The result validates the effectiveness of the graphene/TiO2-coated surface to be utilized for ample time. Hence, this study asserts the durability and robustness of the superhydrophobic graphene/TiO2-coated PLA film and substantiate it to remain functional in the long run.The durability of the graphene/TiOSTM D870 . The rested film . The con2 nanoparticles composite. This study reports the effectiveness of the graphene/TiO2 nanoparticles for fabricating the superhydrophobic surface. The water-repelling effect exhibited by the graphene/TiO2 coating shows a promising antifouling and self-cleaning effect. The strategy herein is proposed as an ideal manufacturing method for superhydrophobic surfaces that are suitable for employment in medical devices. Further investigation is required to study the antibacterial activity, as well as the cytocompatibility, of the graphene/TiO2-coated PLA surface. Nonetheless, this study offers a simple, time-saving, and eco-friendly method of fabricating a durable superhydrophobic graphene/TiO2-coated surface.The PLA films demonstrated superhydrophobic properties upon modification with the graphene/TiO2 with a minimal concentration of 0.5% w/v, via a simple dip-coating method. The graphene/TiO2 coating showed superhydrophobic properties by exhibiting a high apparent contact angle. The apparent contact angle increased with the concentration of nanoparticles. Moreover, the coating of graphene and TiO2 hybrid exerted a synergistic effect by providing an enhanced surface roughness on the PLA surface. It is implied that the structured surface allowed for the entrapment of air pockets, which reduced the direct contact point between the water and graphene/TiO2-coated surface. Therefore, the graphene/TiO2-coated PLA film demonstrated low wettability. Additionally, the ratio of graphene to TiO2 influenced the wettability of the coated surface. In brief, a superhydrophobic surface was achieved by using a minimal amount of graphene and TiO2 nanoparticles. In addition, the graphene/TiO2 coating possesses significant durability and stability by demonstrating low degradation after overnight water immersion. This study shows a promising nanoparticles composite, prepared with graphene and TiO2, that is suitable for introduction into medical devices, where they can deploy their antifouling properties.A superhydrophobic surface was successfully fabricated by using graphene and TiO


The expression of TDP-43, the main component of neuronal intracellular inclusions across a broad spectrum of ALS and FTD disorders, is developmentally regulated and studies in vivo have shown that TDP-43 overexpression can be toxic, even before observation of pathological aggregates. Starting from these observations, the regulation of its expression at transcriptional level might represent a further key element for the pathogenesis of neurodegenerative diseases. Therefore, we have characterized the human TARDBP promoter, in order to study the transcriptional mechanisms of expression. Mapping of cis-acting elements by luciferase assays in different cell outlined that the activity of the promoter seems to be higher in SH-SY5Y, Neuro2A, and HeLa than in HEK293. In addition, we tested effects of two SNPs found in the promoter region of ALS patients and observed no significant effect on transcription levels in all tested cell lines. Lastly, while TDP-43 overexpression did not affect significantly the activity of its promoter (suggesting that TDP-43 does not influence its own transcription), the presence of the 5′UTR sequence and of intron-1 splicing seem to impact positively on TDP-43 expression without affecting transcript stability. In conclusion, we have identified the region spanning nucleotides 451–230 upstream from the transcription start site as the minimal region with a significant transcription activity. These results lay an important foundation for exploring the regulation of the TARDBP gene transcription by exogenous and endogenous stimuli and the implication of transcriptional mechanisms in the pathogenesis of TDP-43 proteinopathies. After initial implication in the pathogenesis of monosymptomatic forms of cystic fibrosis7, it has been discovered a direct association between TDP-43 abnormalities and neurodegenerative diseases, and in particular with Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Degeneration (FTD)9 was discovered. Over the last years, TDP-43 immunoreactive inclusions have also been reported in 70% of patients with hippocampal sclerosis, 30% of patients with Alzheimer's disease, 33% of patients with Pick's disease and in a subset of patients with Lewy-body related disease17. Whereas many efforts have been expended in characterizing TDP-43 pathophysiological functions, a contentious debate ensued and is still ongoing over the possible mechanisms whereby TDP-43 triggers neurodegeneration. It has been proposed both that cytoplasmic aggregates might be neurotoxic ("gain of function" hypothesis) and that the sequestration of the factors in aggregates might induce a functional deficiency resulting in alteration of the TDP-43-regulated processes ("loss of function" hypothesis)22. Of course, the two hypotheses are not necessarily mutually exclusive, and studies with animal models have shown that TDP-43 overexpression can be neurotoxic, even without presence of inclusions in worms23, flies26, mice33, and monkeys34. Importantly, mice expressing the mutant A315T TDP-43 mice show a dose-dependent degeneration of cortical and spinal motor neurons, without evidence of cytoplasmic TDP-43 inclusions32. These studies suggest that even an increase in TDP-43 can contribute to neurodegeneration without occurrence of aggregation. In addition, elevated levels of TDP-43 has been found in the cerebrospinal fluid37 as well as in plasma of ALS40, FTD and Alzheimer42 patients, and in FTD patients carrying C9orf72 expansion or GRN mutations, plasma phosphorylated TDP-43 levels have been found to be higher than in healthy controls43. On the other hand, it is now well established TDP-43 levels are tightly controlled: in fact, it has been demonstrated that TDP-43 can regulate its own expression through a negative feedback loop51. Altogether these observations suggest that regulation of TDP-43 expression at transcriptional level might also be implicated in the pathogenesis of neurodegenerative diseases.TAR DNA-Binding Protein 43 (TDP-43) is an ubiquitously expressed and highly conserved nuclear protein encoded by the human TARDBP gene, and it is involved in several cellular mechanisms, including transcription, pre-mRNA processing, splicing and translation52. However, no functional studies have been so far carried out.The study of the TARDBP gene promoter is therefore useful to shed further light on the pathogenesis of TDP-43 proteinopathies. Interestingly, two SNPs were found within the promoter sequence of TARDBP gene in ALS patientsIn this work, we have characterized the cis-acting elements important for TARDBP gene regulation and evaluated its cell-type specificity. Subsequently, we have characterized the possible impact of some cis-acting elements (SNPs and 5′UTR), trans-acting factors (TDP-43 itself) and events (splicing) on the transcriptional activity of TARDBP promoter.TARDBP gene sequence (Ensembl gene ID: ENSG00000120948) was initially performed by a bioinformatic analysis using the UCSC Genome Browser (GRCh37/hg19). The TARDBP gene, mapped to chromosome 1p36.22 region, spans 12870 bp from position 11,072,679 to 11,085,548 on the forward DNA strand. Figure http://dbtss.hgc.jp54), most of transcription start sites (TSS) mapped at the corresponding positions indicated in the Reference Sequence NM_007375  failed to detect the main core promoter elements. These observations suggest that the expression of the human TARDBP gene is driven by a TATA-less promoter.Subsequently, by querying the Eukaryotic Promoter Databasehttp://www.ensembl.org/). Alignments were performed using MUSCLE software57. Among primates, we compared the genomic sequences of the putative TARDBP promoter region from Hominoidea , from of New World monkey  and from Old World monkey . In general, this comparison highlighted that the relative degree of similarity among all three species increases significantly with respect to the proximity of the TSS. Then, in the region spanning from nucleotide -1316 to -1000 upstream from TSS, the hominoid sequences seem to be closer to each other than with that of New and Old World monkeys. On the other hand, in the region spanning the 1000 nucleotides upstream from TSS, the sequences from all primates show a high degree of identity , without outlining regions evolutionary conserved between these two species (data not shown).In order to study the evolutionary conservation of TARDBP promoter, the 1316 nucleotides upstream of the TSS of TARDBP transcripts from human and other species were retrieved using GenBank and Ensembl Genome Browser  to 1316, 927, 451, 380, 320, 280, 230 and 180 nucleotides upstream from the TSS  and the luciferase activity of each construct was normalized versus the 1316 construct .Intracellular analysis showed that most of the promoter activity was retained in the fragments spanning from 1316 to 451 nucleotides upstream from the TSS within all tested cell lines  and in HeLa cervical carcinoma cell line than in HEK293 embryonic kidney cells  has revealed two promoter variants (c.1-562t>c and c.1-100t>c) with a different frequency in patients than in controls 52. The c.1-562t>c single nucleotide substitution  was found in homozygosity (C/C) at higher frequency and in heterozygosity (T/C) at lower frequency in sALS patients, as compared to controls . On the other hand, the c.1-100t>c polymorphism  was present only in heterozygosity (T/C) at higher frequency (0.2 vs 0.1) than in controls52.The analysis of pathogenic mutations found in the Therefore, we sought to test the impact of these SNPs on the transcription of TDP-43, in order to find a possible functional correlation with the manifestation of ALS. In order to analyze the SNP effects alone or in combination, we created three variants of our 927 original pGL4 construct , so reproducing all the possible alleles ,  and  Fig. A. The co62 and was used as it allows a more homogenous TDP-43 expression from a Tetracycline inducible expression vector.After characterizing the sequence of the promoter of TARDBP gene and its activity in different cell lines, we wished to evaluate the ability of TDP -43 to influence, directly or indirectly, the synthesis of its own transcript. For transfections of these constructs, a stable HEK293-flp-in cell line carrying a single copy of flag-tagged wild type human TDP-43 (HEK-TDP43wt) was used. This cell line was generated in our laboratoryThe HEK-TDP43wt cells were transfected with the constructs 451, 927, and 1316 . Subsequently, TDP-43 expression was driven by Tetracycline induction (48 h) and its levels were probed by Western blotting  and the first 12 bp of exon 2, separated by intron 1 (972 bp). In order to explore the presence of additional elements able to modulate TDP-43 expression, the functional impact of the 5′ UTR (construct 451 + Ex1Ex2) and intron 1 (construct 451 + Ex1-IVS1-Ex2) of mid Fig. A. A firs63. Considering the higher mRNA levels derived from the intron-containing construct (451 + Ex1-IVS1-Ex2 wt), to prevent the possibility that the decay machinery might be saturated by the spliced mRNAs, we used proportionately less of this construct, while keeping constant the amount of total transfected DNA. While it was not possible to determine the half-life of the 451 + Ex1-IVS1-Ex2 mut mRNA (whose expression was negligible), the mRNA species of both the 451 + Ex1-IVS1-Ex2 wt and the 451 + Ex1Ex2 constructs exhibited half-lives of ∼ 7 h, indicating no significant differences in mRNA stability dependent on splicing  outlined how, in most TDP-43 transcripts, transcription starts from the TSS indicated in the main transcript isoform NM_007375. Regarding the spatio-temporal expression pattern, TDP-43 is a developmentally regulated protein64, while its distribution in adult tissues seems to be ubiquitous64. These observations are consistent with the hypotheses supported by recent studies reporting that TATA box is present in only a minority of promoters67, often with tissue-specificity such as liver and muscle70.We have experimentally characterized the promoter region of the human TARDBP gene. A previous prediction indicated that the core regions of the TARDBP promoter could be located between 500 nucleotides upstream of exon 1 and 66 nucleotides downstream of exon 1 and in two regions of intron 1 (212 and 613 nucleotides)From an evolutionary perspective, it is interesting to note that, among primates, the greatest degree of evolutionary conservation encompasses the 600nts upstream of the TSS. On the other hand, the alignment of human and rodents sequences highlights the 200nts upstream of the TSS as the region of higher homology. These observations suggested that the region in-between plays an important role in directing transcription of the TARDBP gene. Indeed, this hypothesis is consistent with the experimental mapping performed in this study that has outlined the region spanning nucleotides 451–230 as necessary for the minimal promoter activity in all the tested cell lines  cellular toxicity or alteration of the TDP-43-regulated gene expression. Understanding the factors that control TDP-43 expression within cells will certainly provide a better insight into disease origin and progression. Indeed, it has been observed that an increase of TDP-43 protein levels can activate the aggregation process in both cytoplasmic and nuclear compartmentsLooking for potential modifiers of the TARDBP promoter activity, we have focused the attention on two SNPs  previously identified within the TARDBP promoter of SALS patients. Although the variants do not change any transcription factor binding site, we have functionally tested their transcriptional impact. Although, in our system, we did not find statistically significant differences in the promoter activity of each haplotype, we can hypothesize that the potential regulatory SNPs do not lead to variation of TARDBP promoter activity directly, but cannot exclude that other possibilities of how these may affect transcription levels do exist. The variants might affect transcription at endogenous copy numbers, but not for overexpressed exogenous constructs. In addition regulatory elements, outside the promoter region included in our reporter plasmid and might interplay with the variants and modulate the activity of the promoter.75, we verified if there are transcriptional effects of TDP-43 on its own promoter activity and it does not modulate its own transcription.Subsequently, since TDP-43 autoregulates its own expression through a negative feedback mechanism via direct interaction with its own 3′UTR76, we next sought to test the possible effects of the TARDBP 5′UTR and intron 1 splicing on the expression of the associated reporter gene  indicated that this element can positively influence gene expression. Consistently, recent studies have shown that approximately 35% of human 5′UTRs are annotated as harboring introns64, in a cell specific fashion, and, through aging, these fluctuations are evolutionary conserved at least in mice and flies59. Notwithstanding there are no clear data of age-related changes in TDP-43 in human83, altogether these observations suggest that a better understanding of the mechanisms and factors involved in controlling the activity of the TARDBP promoter and, consequently, TDP-43 expression will give clues to uncover novel processes implicated in the onset and progression of the TDP-43 proteinopathies.In conclusion, growing lines of evidence suggest that the pathophysiology of ALS, FTD and other neurodegenerative diseases might be the consequence of the convergence of multiple risk factors acting also on changes of TDP-43 levels. Indeed, TDP-43 expression can be modulated during developmentThe 1316 bp region upstream of the TARDBP TSS was amplified by PCR and cloned in pGEM-T easy vector. The 1316 bp insert was digested with the restriction enzymes KpnI and HindIII (these sites were designed into the primers used in PCR) and subcloned into the pGL4.1 vector  digested with the same restriction enzymes. Other 7 deletion-mutants of TDP-43 promoter  were then generated by PCR using gene-specific forward primers and the pGL4-1316 construct as template. Deletion-mutant inserts were KpnI-HindIII-subcloned in the pGL4.1 vector. TARDBP promoter SNPs were generated by PCR mutagenesis. The identity of all constructs was verified by DNA sequencing. Primer sequences are available.The cell lines used for transfections were the following: HEK293, HeLa, Neuro2A, and SH-SY5Y. Each construct was transfected in duplicate, 600 ng of DNA was diluted in a final volume of 100 µl with Opti-MEM I Reduced Serum Medium . To normalize for transfection efficiency, the cells were co-transfected with 20 ng/well of pRL-TK  plasmid along with the promoter constructs. In parallel, 98 µl of Opti-MEM were added to 2 µl Lipofectamine 2000 Reagent . After 5 min. at room temperature, the two solutions were combined and mixed, and 20 min later, the growth medium was removed from the cells and replaced with fresh DMEM containing 5% FBS. Then, the transfection mix was added to each well.44.Total cell lysate was prepared from cells 48 h post transfection and firefly luciferase activity was assayed using the Beetle-Juice KIT  and a Turner Design 20/20 Luminometer . Control transfections with pGL4.1 empty vectors were used as negative controls. Data presented are mean ± Standard deviation (SD) of at least three independent experiments and given as fold expression overexpression indicated in the Figures set arbitrarily at 1. Statistical analysis was performed using Student’s T or ANOVA tests. In order to verify the possible influence of TDP-43 on its own promoter, we used the previously generated human stable cell line Flp-In HEK293 -TDP-43 wild type, carrying the human TDP-43 transgene locus-specific-integrated, whose expression is driven by an inducible version of the Tetracycline (Tet) promoterOne day before transfection, Flp-In HEK293 -TDP-43 wild type (wt) cells were seeded at 60% confluence. Lipofectamine reagent was used for transfection with 300 ng of the reporter plasmids and 20 ng of pRL-TK vector. After 12 h, the medium was changed, and the cells were incubated further for 24 h. Flag-tagged TDP-43 expression was induced with 1 µg/ml tetracycline. The luciferase/renilla activities were then assayed and statistical analyses were performed using Anova one-way followed by Tukey's test.In order to quantify the mRNA levels of the 5′UTR TDP-43 constructs, co-transfections of luciferase constructs and pRL-TK were carried out as described in the section "Transfections and Luciferase assays".84 and the expression levels were numerically expressed as the ratio of applicant density of the target amplicons over that of GAPDH.Relative expression of constructs 451, 451 + Ex1Ex2, 451 Ex1-IVS1-Ex2 wt and 451 Ex1-IVS1-Ex2 mut constructs was performed by semi-quantitative RT–PCR and by quantitate Real Time PCR. Forty eight hours after transfection, cultures were terminated and RNA was extracted with Trifast reagent , according to manufacturer’s instruction. In the semi-quantitative RT–PCRs, GAPDH was used as internal control. The sequences of primers were (5′–3′): GAPDH_Ex1s, 5′-CGCTCTCTGCTCCTCCTGTT-3′; GAPDH_Ex2as, 5′-CCATGGTGTCTGAGCGATGT-3′; TDP-43 Ex1 + 9 (277 bp)_s, 5′-AGCTTGCGCCATTTTGTGGGAGCGA-3′; Luc ATG + 155(277 bp)_as, 5′-GTAATGTCCACCTCGATATGTGCGT-3′. Optical density of amplicons was calculated using the ImageJ image processing programQuantitation of the 5′UTR TDP-43 mRNAs by Real Time PCR, co-transfections of luciferase constructs and pRL-TK were carried out as described in the section "Transfections and Luciferase assays". The extracted RNA (1 µg) was DNAse-treated and reverse-transcribed using the M-MLV reverse transcriptase , and oligo-dT (0.2 µg/reaction as primer), in a final volume of 40 µl, essentially as indicated in the manufacturer's instructions. The CFX96 real-time PCR detection system  was used for all qPCRs. SYBR green assays were optimized  with the iQ SYBR Green Supermix (Bio-Rad), according to the manufacturer's instructions, using tenfold serial dilutions of cDNAs. The amplification mixture consisted of 0.2 μM primers, 5 μl of SYBR Green Supermix, and 4 μl template DNA in a total volume of 10 μl. Samples were amplified with the following program: initial denaturation at 95 °C for 2 min, followed by 40 cycles of denaturation for 15 s at 95 °C and annealing/elongation for 30 s at 60 °C. The expression of the co-transfected renilla orf was used to normalize the luciferase qPCR data.Primer sequences were: Luciferase (62 bp), forward 5′-CACAAAGCCATGAAGCG-3′ and reverse 5′-ATATGTGCGTCGGTAAAG-3′; Renilla (140 bp), forward 5′-CAGAACAAAGGAAACGGATGA-3′ and reverse 5′-CGCGTTACCATGTAAAAAA-3′.85.Melting curves were examined to determine the specificity of each product and relative expression data were calculated with the ΔΔCT method85.For experiments with actinomycin D, SH-SY5Y cells were transfected with 3000 ng of no-intron constructs  or of the 451 Ex1-IVS1-Ex2 mut plasmid or 600 ng of the 451 Ex1-IVS1-Ex2 mut construct in a p100 plate. Total DNA was normalized to 3000 ng by adding an appropriate amount of pUC18 vector. Twelve hours later, cells deriving from each transfection were harvested and plated in different wells (one for each actinomycin incubation time point). Twenty-four hour after plating, actinomycin D (5 μg/ml) was added. The relative levels of the indicated mRNAs were assessed at the designated time points, following shutoff of transcription using qRT-PCR. Samples were collected at the indicated times using TRIzol. Total RNA from these samples was analyzed via qRT-PCR using the abovementioned Luciferase primers and normalization was carried out versus the housekeeping gene RPII . Relative transcript abundances were determined using the ΔΔCt methodCells extracts were prepared in PBS containing 1 × protease inhibitor . Proteins were separated by SDS-PAGE and transferred to nitrocellulose  and protein detection was carried out with standard Western blotting techniques. After transfer, membranes were incubated for 10 h in blocking solution  to prevent non-specific binding. Subsequently, membranes were incubated for 1 h at room temperature with specific primary antibodies diluted in blocking solution. Expression levels of both endogenous and flag-tagged human wild-type TDP-43 was monitored by using a commercially available polyclonal anti-TDP-43 antibody . Endogenous tubulin was used as a loading control, using an in-house made mouse monoclonal antibody. Immunoblots were developed by using the ECL Star Enhanced Chemiluminescent Substrate .Supplementary Figures.


Low-dimensional van der Waals (vdW) materials can harness tightly confined polaritonic waves to deliver unique advantages for nanophotonic biosensing. The reduced dimensionality of vdW materials, as in the case of two-dimensional graphene, can greatly enhance plasmonic field confinement, boosting sensitivity and efficiency compared to conventional nanophotonic devices that rely on surface plasmon resonance in metallic films. Furthermore, the reduction of dielectric screening in vdW materials enables electrostatic tunability of different polariton modes, including plasmons, excitons, and phonons. One-dimensional vdW materials, particularly single-walled carbon nanotubes, possess unique form factors with confined excitons to enable single-molecule detection as well as in vivo biosensing. We discuss basic sensing principles based on vdW materials, followed by technological challenges such as surface chemistry, integration, and toxicity. Finally, we highlight progress in harnessing vdW materials to demonstrate new sensing functionalities that are difficult to perform with conventional metal/dielectric sensors. This review presents an overview of scenarios where van der Waals (vdW) materials provide unique advantages for nanophotonic biosensing applications. The authors discuss basic sensing principles based on vdW materials, advantages of the reduced dimensionality as well as technological challenges. The real-time, label-free sensing capability of this modality has been critical for the functional and quantitative characterization of antibodies3, drugs, aptamers, target-drug interactions4, and virus-host-cell receptor interactions5. SPR generates invaluable time-resolved kinetics data that cannot be obtained with conventional end-point binding assays that employ target labelings, such as enzyme-linked immunosorbent assays (ELISA) or radioimmunoassays. State-of-the-art SPR instruments can achieve limits of detection (LOD) that are comparable to ELISA and they are increasingly being applied toward the clinical analysis of patient biofluids to detect proteins, microRNAs, drugs, and small molecules associated with various disease conditions6. The refractive index (RI)-sensing transduction mechanism of SPR eliminates the need for labeling and washing steps, provides real-time kinetic information and is fast, which can be advantageous for clinical applications. Furthermore, SPR is useful for characterizing low-affinity analytes, which in equilibrium ELISA assays would require larger sample volumes and cause unwanted dissociation of weakly-bound analytes during wash steps6.Photonic biosensors are indispensable tools for life sciences and medical diagnostics. For example, commercial surface plasmon resonance (SPR)9. While nanoplasmonic structures add fabrication complexity and costs, they can improve multiplexing capacity, miniaturization, and sensitivity while also offering considerable design flexibility. Early demonstrations focused on enhancing the performance of conventional SPR by reducing the footprint of the sensing area to provide portability for point-of-care (POC) applications and/or increase parallel detection for high-throughput screening. For example, the widely used gold nanohole arrays11 can be excited with simple collinear optics compatible with a standard microscope, while simultaneously enabling array-based sensing via parallel imaging techniques12. The challenge of producing large-scale patterns with ease and low cost has been addressed using interference lithography13, replication from templates14, and photolithography15.To miniaturize and further improve the performance of SPR sensors, researchers have leveraged both top-down lithography and bottom-up synthesis to build “nanoplasmonic” sensors by engineering the flat gold films of conventional SPR into nanoparticles, nanoholes, or collections of sub-wavelength unit cells called “metasurfaces” of various shapes. Such nanostructures and metasurfaces can extend resonances to broader frequency ranges , and exhibit optical phenomena such as localized surface plasmon resonance (LSPR), radiative coupling, and extraordinary optical transmission18. By taking advantage of the nanohole geometry, which can selectively localize receptor molecules or biological nanoparticles, researchers have shown unique sensing functions utilizing nanovesicles11  single-walled carbon nanotubes (SWNTs) and two-dimensional (2D) graphene—have already demonstrated novel sensing capabilities that are inaccessible to metal/dielectric nanophotonic sensors. For example, SWNTs have been used for single-molecule detection27  materialsons Fig.  for new n27 Fig.  and in v 28 Fig.  via excig29 Fig. , moleculg29 Fig. , on-chip 30 Fig. , and nan 31 Fig. .Fig. 2LoIn this review, we explore the potentials of vdW materials for nanophotonic biosensing Fig. . We starQ) factor, and are of high contrast. When these conditions are met, analyte molecules can result in large intensity changes in the photodetector  may not be enough to characterize the LOD of disparate biosensors. A more fundamental quantity for nanophotonic structures is the evanescent field decay length of the resonant field into the sensing medium , of bound receptor-analyte pairs by subtracting the rate of dissociation from the rate of association:In this illustrative example, receptor molecules  SARS-Cov and (b) SARS-Cov2) are immobilized on the gold SPR sensor surface and their binding interactions with the target analytes (human ACE2 protein) are measured at different concentrations. A standard equation for 1:1 binding kinetics calculates the change in the normalized surface coverage, ca is the analyte concentration in solution, kon (mol−1 L s−1) is the association rate and koff is the dissociation rate. The equilibrium dissociation constant Kd = koff/kon (M) measures binding affinity (lower Kd means higher binding strength) and the equilibrium surface coverage is Kd) than SARS-CoV (a and b were plotted using the 1:1 binding kinetics equation and parameters from ref. 5), providing quantitative clues to its more infectious nature. (d) An example of reflectance vs. wavelength obtained from a conventional prism-based SPR setup. Conventional prism-based SPR has relatively broad resonance and typically the quality-factor  is on the order of 10. Resonance shift in SPR is measured by either bulk sensitivity (shift per RI change of the bulk solution), or the more biologically relevant thin-film sensitivity (shift per adsorbed film thickness). Conventional prism-based SPR setups exhibit very high bulk sensitivity of up to ~10,000 nm/RIU, or ~100°/RIU2. Resolution is defined as the minimum detectable RI change. This depends on multiple factors such as instrumentation technique, resonator design, and environmental control156. Despite relatively low Q’s, with external controls, robust curve-fitting algorithms, and schemes to reduce photodetector shot noise and source fluctuation, state-of-the-art SPR can resolve spectral shifts as small as 0.001 nm from a resonance dip156, which equates to a resolution of (0.001 nm)// = 10−7 RIU. However, bulk sensitivity does not always correlate with sensitivity to the molecular binding. For example, while nanoplasmonic sensors  (e) typically show lower bulk sensitivity (~700 nm/RIU in this example) than conventional SPR, they utilize more tightly localized evanescent fields and could show similar performance for detecting surface-bound molecules. Conversely, long-range SPPs show higher bulk sensitivity 158 than conventional SPR, but their molecular LOD is similar. A good sensitivity metric for surface-bound molecules can be estimated in terms of picograms/cm2, and thin-film sensitivity (shown in (f)), as measured by resonance shifts with deposited Al2O3 film thickness on gold nanodisks/nanoholes34 is a practical parameter for predicting detection limits159. Using new degrees of freedom available with nanostructures, researchers have engineered high-Q resonances. The figure-of-merit (FOM), defined as (bulk sensitivity)/(linewidth) considers intrinsic/extrinsic plasmon damping mechanisms and can serve as another useful metric. In particular, when an intensity-based sensing scheme is used , a narrower peak results in a greater intensity change at a fixed wavelength per RI change. The FOM can be increased by engineering structural resonances  to reduce the resonant linewidths. However, such changes also tend to reduce the intensity of the resonance, thereby degrading the overall sensitivity of detection. Likewise, arguments based on Q or FOM alone would favor high-Q dielectric resonators over metal plasmonics, but their molecular detection sensitivities are relatively similar. This is due in part to the low contrast in signal, determined by the relative magnitude of the resonant peaks. For continuous monitoring of binding kinetics at high temporal resolution (~millisecond acquisition time), it is essential to deliver high photon flux at the photodetector, which requires that the resonance dip or peak should be large in magnitude .where 5 and the 1:1 binding kinetics equation. (d) Example of SPR reflection spectrum from a prism-based setup. (e) Resonance shift measured for a high-quality-factor subradiant SPP mode with changing NaCl concentration. Reprinted with permission from ref. 157. (f) The resonance shift vs. ALD-grown Al2O3 film thickness was measured from which one can obtain the LSPR field decay length. Adapted with permission from ref. 34.Illustrative examples of SPR binding kinetics for purified recombinant receptor-binding domains of viral spike protein from (a) SARS-CoV and (b) SARS-CoV2, against human ACE2 protein plotted using (c) association and dissociation rates from ref. 37. While the surface of gold SPR sensors is commonly functionalized via a self-assembled monolayer of alkanethiols, the surface of graphene or other vdW materials requires different strategies for functionalization, which presents challenges as well as new opportunities. Graphene surfaces can be modified by attaching some molecules via π-stacking or other functional groups . However, the ease of attachment via π-stacking also implies increased nonspecific binding of interferents from biological samples, necessitating a proper blocking procedure. Xue et al. used antimonene-modified gold SPR chips to detect microRNA hybridization events31. Density functional theory energetic calculations predicted that antimonene surfaces have a higher affinity for single-stranded (ss) DNA than double-stranded (ds) DNA. By using gold nanorods to further boost SPR signals, they were able to detect ssDNA-microRNA hybridization events. vdW materials can also protect the surfaces of reactive metals—especially copper and silver—which is necessary for SPR biosensing applications38. Wu et al. characterized the potential of layer materials  to protect the chemical and optical properties of plasmonic metals39 At a wavelength of ~1 µm, they achieved bulk RI sensitivity of ~10,000 nm/RIU, which is comparable to the sensitivity of conventional spectroscopic techniques.Building on the mature capabilities of prism-coupled gold-film SPR instruments, researchers have explored the benefits of coating the gold film with various 2D materials. Groups have reported improved SPR sensitivity from graphene-coated gold surfaces, which was attributed to enhancement of the surface electric fields provided by an added charge transferred from graphene to goldWhile these studies demonstrate impressive LOD for molecules in buffer, the response of hybrid vdW-metal surfaces in biofluids for practical biosensing applications  needs to be characterized to ensure whether the same low LOD can be maintained. Also, instead of single-channel measurements, reference-channel measurements, as well as temperature stabilization, will be essential for ultrasensitive detection.40. However, the large-area growth and transfer of vdW materials atop metal films add complexity, labor, and costs. As such, in order to justify their use in practical biosensing applications, the benefits need to go beyond incremental gains in sensitivity and enhanced surface adsorption of analytes. Ideally, vdW nanophotonic biosensors should be used in tandem with other sensing modalities, such as electrical or electrochemical detection, or to enable novel functionalities such as vibrational fingerprinting with spectroscopy.As shown in these examples, the applications of vdW materials for conventional SPR or other nanophotonic RI sensors are rapidly expandingRefractometric sensors made with metals, dielectrics, and metal-vdW hybrids have shown impressive performance metrics, but cannot identify bound analyte molecules. Vibrational spectroscopy techniques like infrared (IR) absorption spectroscopy and Raman scattering complement SPR and enable such “fingerprinting” functions for molecules, viruses, extracellular vesicles, and cells. With their electrically tunable doping and ultra confined mid-IR and terahertz (THz) plasmons, graphene and other 2D vdW materials are especially promising for these applications.−1). IR spectroscopy taps into this so-called fingerprint region and is used in numerous scientific fields  and in the industry . However, the large mismatch between micrometer IR wavelengths and nanometer-sized molecules leads to low sensitivity, limiting the use of IR spectroscopy for measuring trace amounts of samples. Nanophotonics can bridge this gap by producing strong, tightly localized near-fields in the vicinity of resonant optical nanostructures. This approach is called SEIRA  typically exhibit lower enhancement factors (EF) of ~10nts Fig. , which dses Fig. . They sh 50 Fig. .Fig. 3De51. For instance, while metals can support strong plasmonic responses due to their high electron concentration, they suffer from high loss and provide resonators with low Q-factors . In contrast, Mie resonances in low-loss and high-index dielectric metasurfaces can achieve several orders of magnitude higher Q-factors. Tittl et al. used CMOS-compatible high-Q-factor (>100) silicon resonators in a sensing approach that can convert mid-IR molecular fingerprints into chemical-specific barcode-like images while eliminating the need for bulky IR spectroscopy instruments52. Leitis et al. used germanium metasurfaces to expand the spectral coverage of resonant SEIRA over 1000 cm−1 wavenumber and extracted vibrational signatures of different molecules in a multi-step assay53.To date, SEIRA has mostly employed metal-based plasmonic resonators with engineered nanostructures in various shapes and array configurations. However, this landscape is changing rapidly, with novel nanomaterials that can address the intrinsic shortcomings of metals54. The unique optoelectronic properties of graphene enable external control over plasmonic resonance frequency by changing its carrier density  with electrostatic biasing57. Such active tuning is used to realize dynamically configurable IR sensors tuned to specific molecular vibrational modes. Rodrigo et al. showed the first graphene-based tunable mid-IR biosensor and its potential for quantitative chemical-specific detection26. They measured plasmon resonance spectral shifts accompanied by narrow dips corresponding to the surface-enhanced molecular vibration bands of a protein analyte over a broad spectrum . Graphene nanoribbons can provide up to two orders of magnitude higher field confinement than metallic plasmonic dipole antennas, and thus enable unprecedentedly strong overlap of light with nanometric biosamples26  is typically used to synthesize large-area graphene, and the resulting grain boundaries, impurities and defects result in lower quality graphene, as shown by its reduced carrier mobility (~1000 cm2 V−1 s−1). Furthermore, nanopatterning of graphene with small structures  is often required to excite mid-IR plasmons, but the rough edges induced by the nanofabrication process lead to additional loss and further deteriorate the quality of the resonators. Limited plasmon lifetimes (~50 fs) and inelastic scattering with optical phonons have also been reported in graphene nanoribbons67. However, further effort is needed towards the development of high-quality 2D vdW materials that can be scalably manufactured for real-world applications. Observation of low-loss graphene from the encapsulation of exfoliated samples with hexagonal boron nitride (hBN)68 and recent advances in CVD graphene transfer involving proper cleaning and encapsulation, which result in room-temperature mobility in excess of 10,000 cm2 V−1 s−1, demonstrate some of the important ongoing progress in materials development69.Graphene can achieve high Q-factors, which can lead to increased sensitivity due to longer interaction time between the probing IR light and the analytes. In theory, Q-factors well over 100 can be expected with exfoliated grapheneEf), but this requires some care in terms of fabrication and design. In the past, devices typically relied on a thick dielectric spacer between the graphene and the gating layer. This leads to low capacitance values, and hence requires the application of high bias voltages (tens of volts). However, recent research shows that incorporation of a field-effect transistor (FET) device architecture and suitable spacer films as a substrate can greatly reduce the bias voltage while enabling high-speed operation and incorporation of imaging70. A related issue stems from the dependence of the graphene response on the optical properties of the spacer layer. Graphene plasmons can strongly couple with the phonon modes of the substrate, and this hybridization can produce complex spectral features and limit the operating spectral window. Hu et al. incorporated CaF2 nanofilms as spacers; since CaF2 has no phonons in the relevant range, it eliminates this problem and allows the use of SEIRA over a broader spectral range59.Dynamic tuning of graphene plasmons see Box  is achie71, integration with photonic cavities 72, and hybrid substrates incorporating plasmonic nanostructures73 , which is not practical for device applications. The coupling efficiency is further hampered by the low carrier density and the weak oscillator strength of graphene plasmons. In order to excite stronger plasmonic responses, recent works have used multi-layer stackss73 Fig. . Strong s73 Fig.  can also74. Because the field intensity decays exponentially from the plasmonic substrate surface with a decay length of ~100 nm, one can selectively probe surface-adsorbed molecules and minimize interference from water molecules in the sample. In contrast, graphene resonances are extracted from either the extinction spectra in the transmission configuration or the absorption spectra obtained in the reflection configuration with an optically thick metal layer, which make in situ measurements not possible. Recent work has combined graphene with attenuated total reflection (ATR) prisms for in situ studies, but these approaches involve inherently bulky instrumentation and are not suitable to exploit tunable and strongly confined plasmonic resonances76. Graphene and other liquid-impermeable vdW materials offer alternative routes to avoid water absorption by trapping water in nanometric volumes. This approach has been successfully demonstrated with tip-enhanced IR absorption spectroscopy using a graphene liquid cell50, and in graphene-based nanofluidics77.To benefit from these additional functions, graphene-based SEIRA devices need to be compatible with in-solution measurements and microfluidics. To this date, most SEIRA experiments have been conducted in a dry medium due to the high absorption of water and polymers used for microfluidics in the IR spectrum. These constraints are circumvented for metal-based SEIRA by utilizing strong plasmonic confinement and performing plasmon excitation and signal collection in the reflection configuration with an IR-transparent substrate81. The resonances of hBN, however, are spectrally limited in biosensing applications due to the fixed Reststrahlen bands—where the energy band between the longitudinal and transverse optic phonon frequencies Re(ε) is negative and surface phonon polaritons can be excited—which are not aligned with the amide absorption bands of proteins. However, recent work has shown that the Reststrahlen band of MoO3 can be tuned82, which may be useful for chemical sensing applications, although such tuning is not suitable for biomolecules such as proteins and nucleic acids.Besides graphene plasmons, phonon-polaritons in hBN have been utilized for confining mid-IR lightBox 2. Surface-enhanced infrared absorption spectroscopy (SEIRA)Raman scattering and IR absorption are commonly used to obtain molecular structural information. Raman spectroscopy is based on the inelastic scattering through which a molecule either gains energy from (Stokes process) or loses energy to (anti-Stokes process) incident photons. Transitions involved in IR absorption versus Raman scattering processes are subject to different selection rules. Thus, the measured spectra show different and complementary sets of peaks, and their utility depends on the specific application.6–1010 photons striking a molecule are scattered inelastically. Typical non-resonant Raman scattering cross-sections (σRaman) are 10−31~10−29 cm2/molecule—much smaller than for IR absorption (~10−20 cm2/molecule). However, a key advantage of Raman spectroscopy for biosensing is the small scattering cross-section of the water, which minimizes unwanted background in aqueous samples. In contrast, absorption of mid-IR radiation in water can obscure absorption signals from analytes.Raman scattering is extremely weak, as only 1 in 10E| is roughly |E|4, plus a chemical EF of 10–100-fold due to the modified polarizability of molecules on a metal surface, whereas SEIRA increases only with |E|2. In most SERS substrates, the spatial distribution of intense “hotspots” are random, thus the location of these spots cannot be easily predicted for continuous activation. In contrast, SEIRA EFs, while lower, can be more uniformly distributed and predictable. Importantly, the utility of SERS diminishes for probing large biomacromolecules or complexes, because SERS EF drops rapidly over a few nanometers from the metal surface. In addition, intrinsic SERS detection of protein molecules or changes in their orientation/conformation is difficult for large proteins. Autofluorescence can also easily mask weak Raman signals. Resonantly-excited plasmons in SEIRA substrates typically exhibit lower EFs but provide a wider probing range suitable for sensing the adsorption of larger targets.The scaling factor for electromagnetic enhancement for SERS as a function of electromagnetic near-field amplitude |ADev and A0 denote measured optical absorption from a SEIRA substrate and reference device, respectively, with VDev and V0 being the effective volumes where optical fields and target molecules overlap. Infrared reflection-absorption spectroscopy (IRRAS) is a common method to obtain reference signals from a thin film of target biomolecules. In this case, SEIRA EF can be rewritten as:AIRRAS and θ are measured absorption from IRRAS and the incident angle of IR radiation, respectively. The oblique incidence of IR radiation and the dipole-mirror effect is accounted for by ADev is calculated from the power dissipation by integrating The performance of a SEIRA substrate is represented by the EF, which measures the ratio of absorption from a substrate to that of a reference sample when normalized to an effective volume. This performance metric is given as:Box 3. Characteristics of tunable graphene plasmonsGraphene plasmons offer the unique advantages of tight field confinement as well as electrical tunability of plasmon resonances. Plasmons in 2D vdW materials represent the ultimate limit in terms of sub-diffraction field confinement. As shown in the left panel, graphene plasmons can be conceptualized as an extension of short-range SPP waves in a metal film with symmetric charges across the film, and these plasmons are further squeezed as the film is thinned down. The dispersion of conventional graphene plasmons scales as EF is the graphene Fermi level, εeff is the effective dielectric constant of the environment, and q is the momentum. By placing graphene in proximity to a metal plate, mirror image charges are created that modify the plasmon field distribution. The dispersion for the resulting acoustic graphene plasmon now becomes linear . On the other hand, the plasmon resonances of metallic nanostructures are fixed upon fabrication due to the high carrier concentration  for noble metals. The 2D semi-metallic nature of graphene allows for electrical tunability that is not possible with conventional metals. Free carriers in graphene obtained through electrical doping can number 0.001–0.01 per atom, and the corresponding graphene plasmon resonance resides in the mid-IR (3–20 µm wavelength) and THz frequency ranges, which are ideally suited for reconfigurable vibrational spectroscopy with dynamically tunable resonances26.These dispersions assume the same graphene doping with Box 4. vdW materials for trapping of biomolecules via DEP161 DEP, a radiofrequency (kHz–MHz) analog of the optical trapping process, has been used to manipulate biological particles and can be useful in the context of vdW materials. With a relatively simple apparatus (electrodes and a function generator), DEP can be employed to attract, trap, or repel neutral particles based upon their polarizability. When a particle of radius R and complex permittivity 162.Rapid transport of target analytes to the nanophotonic sensor is critical for detecting low-concentration analytes. Nanophotonic sensors typically exhibit small detection areas, and active transport and concentration of analytes can greatly improve the detection speed and overall sensitivity.dx, it is possible to boost its magnitude by shrinking the critical dimensions of the system  without using high bias voltages or electric fields that are detrimental to biological systems (a). Sharp tips163, nanometric gaps, or nanopores164 in metal films have been used for a wide range of DEP applications such as particle filtering, sorting, trapping, levitation, and rotation162. Atomically sharp vdW materials are uniquely capable of creating ultra-strong DEP forces in the context of (b) sharp electrode tips realized with SWNTs166 and (c) graphene-edge DEP169. Barik et al. showed that patterned edges of monolayer graphene can generate high-gradient fields to trap nanoscale objects and molecules with enhanced trapping forces168. Such edge-trapping DEP devices can be made from graphene or other conductive vdW materials patterned over a metal electrode, separated by a thin insulator. Using this setup, polystyrene particles were trapped on the edges of graphene at voltages as low as 0.5 V. Furthermore, both long (10 kb) and short (500 bp) DNA strands were trappable at low voltages (~2–3 V).where The ultra-strong gradient forces at nanoscale distances in graphene-edge DEP have the potential for nanophotonic as well as nanoelectronic biosensing applications, where analytes such as antibodies, proteins, viruses, nanoparticles, or cells could be trapped rapidly using relatively low (~1 V) bias voltages. Furthermore, the electrical readout capabilities of some vdW materials could also enable a path toward hybrid optical/electrical DEP-enhanced sensors. Surface functionalization of the sensor could be utilized to further enhance detection selectivity. DEP can also be combined with rapidly advancing nanopore sensing technologies (d) based on vdW materials, as discussed in the Supplementary Information.(a) Dielectrophoresis is a scalable particle trapping mechanism. Smaller electrode separation and sharper electrode tip can boost field gradients. (b) Carbon nanotube electrodes (adapted from ref. 166 with permission), (c) graphene edge electrodes (adapted with permission from ref. 168), and (d), metallic nanopores (adapted from ref. 164 with permission), can generate high gradient fields at relatively low trapping voltages toward trapping nanoparticles and biomolecules.83. Initially, using metal plasmonic elements, researchers have already addressed some of the challenges. Schwarz et al. demonstrated a compact mid-IR chemical sensing platform84, which monolithically integrates a bi-functional quantum cascade laser/detector coupled to a dielectric-loaded gold SPP waveguide  waveguide-integrated black phosphorous detectors93, and heterogeneously-integrated Si waveguides on mid-infrared compatible substrates, which could extend the detection limit out to 8 μm94. These advancements in miniaturization due to waveguide-mediated delivery of light onto the vdW sensor, could eventually eliminate the need for expensive and bulky FTIR spectrometers as well as laborious alignment of free-space optics via moving mechanical parts.Tightly-confined excitons in graphene and other vdW materials offer additional exciting opportunities for nanophotonic biosensing. One long-term vision shared by many researchers is to realize compact nanophotonic “lab-on-a-chip” devices that integrate light sources, low-loss dielectric waveguides, nanophotonic sensors, and on-chip photodetectors, as illustrated in Fig. ide Fig. . Waveguiide Fig. 85. Incorrus Fig. 87, have 96 can address an entirely new set of problems—such as in vivo nanophotonic sensing and bioimaging. Shortly after the discovery of SWNTs’ near-infrared (NIR) fluorescence, this phenomenon was recognized as ideal for optical imaging and sensing97. The unique electronic properties of SWNTs that result in their bandgap fluorescence have been reviewed by Weisman98. Briefly, SWNTs can be thought of as single sheets of graphene rolled into cylindrical geometries. Their electronic—and thus photophysical—properties depend on the direction of rolling, which can be specified by lattice vectors n and m  chiral species is metallic or semiconducting. The nanoscale-cylindrical shape of semi-conducting SWNTs confines electrons in one dimension along its circumference, leading to what is known as van Hove singularities in the electron density  SWNTs102. The diffusion length is limited by the presence of defects in the nanotube or the supporting substrate. Fluorescence occurs when the exciton relaxes back down to the band edge and emits a photon with energy equivalent to the exciton energy. SWNTs are commonly interrogated by exciting through a range of visible light in the range of the E22 transition, and their NIR E11 emission is then collected. Different semiconducting chirality species with different bandgaps/exciton energies can then be visualized using a 2D excitation-emission profile 107, sodium dodecylbenzene sulfonate (SDBS)110, and sodium cholate (SC)112, and/or enthalpy-driven interactions such as π−π bonding, hydrogen bonding , or ionic interactions between the SWNT surface and dispersants such as aromatic polymers, cationic polymers, and block polymers.Due to intrinsically strong vdW interactions, SWNTs tend to stick together as bundles and do not naturally disperse in an aqueous solution or biological media. Covalent and non-covalent surface modifications of SWNTs are used to promote solubility. Covalent modification is achieved by chemical reactions on the nanotube surface to introduce carboxyl, amine, sulfhydryl, or other functional groups Box 103. Whil28 has been developed to achieve selective recognition of specific molecules les Fig. . In CoPh123. Cognet et al. have observed single proton and diazonium salt binding and release from SDBS-functionalized SWNTs by detecting stepwise changes in fluorescence124. This approach was extended by Zhang et al.115 to detect nitric oxide with SWNTs deposited onto functionalized glass sides, and by Jin et al.125 to detect H2O2 with SWNTs embedded in type 1 collagen thin films. During measurements, each SWNT serves as an individual optical probe, and individual stepwise changes in sensor states correspond to single-molecule binding or release events. The observed bulk SWNT fluorescence is a combination of these single-molecule interactions, and the bulk concentration is obtained through hidden Markov modeling125. The collagen-based SWNT sensors have also been applied to detect real-time H2O2 generation with high spatial resolution from individual live A431 human epidermal carcinoma cells stimulated with epidermal growth factor117  Patterned nanophotonic structures typically utilize heterogeneous materials and interfaces . By conformally coating such structures with a thin SiO2 film via sputtering or ALD, one can make a homogeneous surface compatible with well-established silane chemistry or vesicle fusion to form a lipid bilayer membrane. Reprinted from ref. 171 with permission.172.While ALD-grown oxides have been used for surface modification of metal/dielectric nanophotonic substrates, nucleation of ALD of oxides on graphene is difficult due to graphene’s hydrophobic nature and lack of out-of-plane bonds. Several strategies have been utilized to overcome this limitation, including using physisorbed ozone, hydrogen plasma functionalization, organic self-assembled monolayers, and evaporated metal or oxide seed layers. Such strategies have proven successful, provided that a pristine graphene surface can be achieved174, nanovesicles, or (c) a pore-spanning lipid bilayer (Reprinted from ref. 175 with permission). In general, SLBs are formed directly on a solid substrate with a thin layer of water (~1 nm) between the membrane and the substrate. It is critical that the sensor material  does not adversely affect the conformation or functionality of membrane-bound molecules. Recent work has combined SLBs, lipid nanovesicles, and synaptic vesicles for SEIRA sensing47. Importantly, lipid bilayer membranes can also help prevent nonspecific adsorption of proteins onto the sensor surface. (d) The recently-developed solvent-assisted lipid bilayer (SALB) method can be used to form lipid bilayer membranes directly on bare gold surfaces without a SiO2 layer. SALB can be useful for SPR, SERS, and SEIRA, and is compatible with some vdW materials. Reprinted from ref. 176 with permission. For substrates or receptors that are not amenable to these methods, other options are available, such as the use of membrane-derived particles as biomimetic receptors. Such cell-derived materials may be directly dispersed as nanoparticles on the surface, or physically inserted into patterned substrates via a ‘squeegee’ method177.For applications in membrane biology and drug discovery, it is desirable to interface nanophotonic sensors with highly fluidic lipid bilayer membranes or nanovesicles that can incorporate a rich group of membrane-bound receptors . Membrane proteins are not easily manipulated in solution and do not fold properly outside the membrane, but researchers have used various schemes to address these challenges using a supported lipid bilayer (SLB)103. (f) The surface of graphene can be modified via different strategies, including non-covalent functionalization (π-stacking), intercalation of molecules between graphene and a substrate, and covalent chemical modification of graphene. Image credit: Kim Daasbjerg. (g) Illustration of surface modification and blocking strategies for graphene-coated Cu films for SPR biosensing. Reprinted with permission from ref. 39.(e) Covalent and noncovalent functionalization schemes for SWNTs. Reprinted with permission from ref. 2 surfaces. Graphene is normally hydrophobic in its pristine state, which hinders SLB formation. Interestingly, graphene is also known to exhibit “wetting transparency” to the underlying substrate178. For example, an SLB can be formed via vesicle rupture on graphene on top of a hydrophilic silica surface. In contrast, graphene on gold, which is hydrophobic, leads to the formation of an intact vesicle layer. Indeed, recent work showed that a lipid monolayer, bilayer, or layer of intact vesicles could be formed on graphene depending on the surface treatment179. Biological interfacing of vdW materials with soft lipid membrane interfaces can add unique options to augment the functionality of nanophotonic biosensors, and further research is highly desirable.(h) SLB formation on graphene is not as clearly understood as for gold or SiO130. Label-free optical SWNT sensors are preferable to traditional POC detection methods such as ELISA in that they do not require additional processing steps after sample collection and allow transient data collection131. One example is a fluorescent SWNT-based microarray for detecting real-time protein-protein interactions132. Here, chitosan-wrapped SWNTs are conjugated to nitrilotriacetic acid (NTA) groups that can chelate Ni2+. The NTA-Ni2+ complex then binds to a His-tag on the target proteins, and the binding event is detected by the reduction of SWNT fluorescence. This platform has been further engineered to monitor 1156 capture protein-protein analyte interactions in a signaling network important in apoptosis. Using a different molecular chelator, Dong et al.133 conjugated immunoglobulin-binding proteins to SWNT and detected IgG, IgM, IgG2a, and IgD. Capture protein-modified sensors can also be printed onto a microarray that examines cross-reactivity, competitive, and nonspecific binding of analyte mixtures134. Such optical nanosensors have great potential to advance real-time, multiplexed biomarker detection for disease diagnostics.SWNT-based sensors have also been developed to detect disease biomarkers at low concentrations, with potential application to POC diagnostics, enabling timely treatment at a reduced cost136. A central question that limits biological applications relates to the biocompatibility of SWNTs. Their toxicity has been evaluated in vitro, with a variety of factors including chirality, length137, synthesis method138, SWNT wrapping140 and cell type141, all affecting biocompatibility. SWNTs are a broad class of materials with varying dimensions, functionalization, and formulation, and since many aspects of a given preparation can affect biological responses142, it is important to evaluate toxicity on a case-by-case basis. In addition, response in cell culture might not always reflect in vivo tissue effects, where cell populations are far more heterogeneous, and materials might also be modified once introduced into the body. Thus, it is crucial to test SWNT sensors in vivo, under conditions reflecting the intended route of administration, site of action, and dose.The unique optoelectronic properties of SWNTs are also ideal for in vivo biosensing applications. SWNTs fluoresce within the NIR window (900–1500 nm), where scattering is reduced and tissue absorption is minimal, allowing SWNT fluorescence to penetrate through deeper layers of tissue relative to visible light7-wrapped SWNTs via tail vein injection to detect transient inflammation, using nitric oxide as a signaling marker in mouse liver. The authors demonstrated long-term nitric oxide monitoring by subcutaneous implantation of alginate hydrogel-encapsulated SWNT sensors. The sensor fluorescence signal remained stable for over 400 days, and histological analysis of the implantation sites showed negligible adverse response116. Harvey et al. loaded DNA-wrapped SWNT sensors into a semipermeable membrane and implanted these into the intraperitoneal (IP) space in mice. The sensors successfully detected oligonucleotide hybridization in vivo, and the mice exhibited no obvious behavior changes143. SWNT sensors have also been used to detect lipid concentrations in vivo in the mouse liver144. Galassi et al. injected DNA-wrapped SWNTs through the tail vein in mice, which localized to the liver144. These DNA SWNTs showed a wavelength shift in response to changing lipid concentrations, and once the mice were administered a high-calorie diet, the resulting fat accumulation within the liver could be detected and used to determine the onset of fatty liver disease. SWNTs implanted in the liver maintained the ability to shift wavelength in response to their analyte over a period of three months, showing the stability of such sensors.SWNTs have been widely applied in animal models. Iverson et al. delivered ss(AAAT)145. These sensors detected the ovarian cancer biomarker human epididymis protein 4 (HE4) from both exogenous administration and endogenous production. DNA-wrapped SWNTs encapsulated in poly(ethylene glycol) diacrylate hydrogels have also been used in various marine organisms for biologging purposes, with minimal changes in tissue architecture for catfish, shark, and eel as detected by high-resolution ultrasound146. These examples show that SWNT sensors of various designs have the potential to function safely in vivo.Williams et al. encapsulated antibody-conjugated/DNA-wrapped SWNTs into a dialysis bag and implanted them into the IP space of miceWe have explored nanophotonic biosensing technologies enabled by low-dimensional vdW materials. With continually improving large-scale synthesis methods for vdW materials, high-resolution lithography, and ultrasensitive detection schemes, it may soon be possible to harness these versatile materials for practical POC biosensing applications. Further in the future, this technology could grow to enable previously inaccessible applications such as label-free monitoring of protein conformational dynamics, flexible and wearable biosensors, and in vivo nanophotonic biosensors. Such real-time in vivo sensors could potentially provide quantitative measurements of biological networks, and in humans, could allow prolonged data collection for early detection of disease, or even be coupled to drug delivery devices to enable closed-loop disease control—analogous to the ‘artificial pancreas’ systems that have been developed for continuous glucose control in diabetes. We also expect vdW materials to benefit other functions beyond conventional sensing that will still contribute to better biosensors, such as harnessing vdW materials for on-chip liquid manipulation , high-gradient-field particle trapping, or building waveguide-integrated light sources and photodetectors.Research into graphene and 2D quantum materials for SEIRA sensing is still in its infancy. In the near future, we will likely witness innovations combining high-quality 2D materials with smart choices of substrates and device configurations. These efforts will undoubtedly improve the performance and expand the versatility of SEIRA-based sensors for a wide range of applications.Another exciting strategy for accessing physical and chemical information entails the physical confinement of individual analyte molecules in a configuration with lower degrees of freedom. One such detection modality is based on the confinement of molecules into structures fabricated in the matrix of the 2D material, such as nanoscale apertures or nanopores (Supplementary Note 1).147, and low-loss dielectric waveguides will present unique opportunities for future innovation. Two-dimensional TMDCs have demonstrated strong exciton resonances in optical spectra148, and recent work has shown that the presence of molecules can transform dark states into bright excitons, potentially enabling unambiguous detection of the adsorbed molecules. Excitons in 2D TMDCs can also be tuned by various approaches149. In addition, integration with birefringent and chiral metasurfaces e.g., chiral plasmons or twisted graphene layers151 or valley-dependent chiral coupling in 2D TMDCs152 for measuring chiro-optic response could advance enantioselective biochemical sensing applications153.The library of vdW materials and heterostructures is rapidly expanding to offer customizable choices for fabricating unique nanophotonic biosensors. For SEIRA and other biosensing applications, we envision that hybrid substrates integrating graphene, polar materials, vdW heterostructures, metal antennas, high-Q dielectric metasurfaces155. Ultimately, vdW materials may empower researchers to realize one of the overarching ambitions of nanophotonics—seamless integration of photonics and electronics in the field of biosensing.While this review focused on nanophotonic sensors, electronic and electrochemical sensors based on vdW materials have also been extensively investigatedSupplementary Information