ABSTRACT
In this work, we demonstrated a single molecule photobleaching-based strategy for the ultrasensitive detection of adenosine. A modified split aptamer was designed to specifically recognize individual adenosine molecules in solution. The specific binding of dye-labeled short strand DNA probes onto the elongated aptamer strand in the presence of adenosine resulted in a concentration-dependent self-aggregation process. The degree-of-aggregation (DOA) of the short DNA probes on the elongated aptamer strand could then be accurately determined based on the single molecule photobleaching measurement. Through statistically analyzing the DOA under different target concentrations, a well-defined curvilinear relationship between the DOA and target molecule concentration (e.g., adenosine) was established. The limit-of-detection (LOD) is down to 44.5 pM, which is lower than those recently reported results with fluorescence-based analysis. Owing to the high sensitivity and excellent selectivity, the sensing strategy described herein would find broad applications in biomolecule analysis under complicated surroundings.
Subject(s)
Adenosine/analysis , Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Carbocyanines/chemistry , DNA Probes/chemistry , Fluorescent Dyes/chemistry , Fluorescence , Limit of Detection , Optical Imaging/methods , PhotobleachingABSTRACT
Exploring the diffusion dynamics of a viral capsid proteins (VCP)-functionalized nanocarrier on a living cell membrane could provide much kinetic information for the better understanding of their biological functionality. Gold nanoparticles are an excellent core material of nanocarriers because of the good biocompatibility as well as versatile surface chemistry. However, due to the strong scattering background from subcellular organelles, it is a grand challenge to selectively image an individual nanocarrier on a living cell membrane. In this work, we demonstrated a convenient strategy to effectively screen the scattering background from living cells for single-particle imaging with a polarization-resolved dual-channel imaging module. By taking advantage of the polarization of anisotropic gold nanoparticles (gold nanorods, GNRs), the signals from cell components could be counteracted after subtracting the sequential images one by one, while those transiently rotating GNRs on the cell membrane still exist in the processed image. In contrast to the previously reported methods, this method does not require a complicated optical setup alignment and sophisticated digital image analysis process. According to the single-particle imaging results, the majority of VCP-GNRs were anchoring on the cell membrane with confined diffusion. Interestingly, on further inspection of the diffusion trajectories, the particles displayed anomalous confined diffusion with randomly distributed large walking steps during the whole track. Non-Gaussian step distribution was noted, indicating heterogeneous binding and desorption processes on the cell membrane. As a consequence of the robust background screening capability, this approach would find broad applications for single-particle imaging under a noisy environment, e.g., living cells.
Subject(s)
Capsid Proteins/analysis , Circoviridae Infections/virology , Circovirus/chemistry , Gold/chemistry , Hepatocytes/virology , Metal Nanoparticles/chemistry , Optical Imaging/methods , Anisotropy , Circoviridae Infections/pathology , Equipment Design , Hep G2 Cells , Hepatocytes/pathology , Humans , Microscopy/instrumentation , Microscopy/methods , Optical Imaging/instrumentationABSTRACT
A colorimetric assay is described for acrylamide (AA). It is based on color changes induced by an increase in the distance between gold nanoparticles (AuNPs) that is caused by AA copolymerization. First, AuNPs were modified with a thiolated propylene amide poly(ethylene glycol) that also contains the AA functionality. The carbon-carbon double bonds on the modified AuNPs can be polymerized under the catalysis of a photoinitiator and under UV irradiation. This results in the aggregation of the AuNPs and in a color change from red to gray. In the presence of AA, the distance between the AuNPs increases due to copolymerization with AA, and the solution of AuNPs preserves its original red color. Under optimized conditions, the absorption ratio (A525/A740) of the solution increases linearly in the 1 nM to 10 µM free AA concentration range, with a 0.2 nM limit of detection. Hence, the method meets the need for rapid monitoring of trace AA in food. The method has a relative error (RSD) that is lower compared to the accepted HPLC method. Graphical abstract Schematic of a novel colorimetric strategy for acrylamide (AA) detection based on the increase of distance between gold nanoparticles (AuNPs) caused by AA participated polymerization.
ABSTRACT
Inhibiting the formation of acrylamide (AA) and hydroxymethylfurfural (HMF) during food heating processes has attracted considerable investigative efforts due to potential health concerns associated with these compounds. The main purpose of this work is to demonstrate a strategy to simultaneously inhibit the formation of AA and HMF with sodium glutamate microcapsules selected to confirm the efficacy of this strategy. An asparagine-glucose aqueous model system was prepared containing free sodium glutamate and sodium glutamate microcapsules. Compared to adding free sodium glutamate, the maximum inhibition efficiency for AA and HMF was found to increase by addition of sodium glutamate microcapsules to 19.07 and 84.32%, respectively. Moreover, the kinetics of AA and HMF formation were studied in this model system. The AA inhibition efficiency significantly increased from 6.75 to 60.35% and the HMF inhibition efficiency significantly increased from 5.98 to 79.72% with increasing the reaction time from 25 to 40 min, indicating that the sodium glutamate microcapsules strategy proves to be far superior at prolonged heating times. These findings suggested that this inhibition strategy may provide promising characteristics for a variety of applications in food processing.
ABSTRACT
Herein, a novel fluorescent/colorimetric/photothermal biosensor is proposed for aflatoxin B1 (AFB1) detection in food based on Prussian blue nanoparticles (PBNPs) (â¼50 nm), gold nanoclusters (AuNCs), and an aptamer (Apt) within three hours. Briefly, a multifunctional compound, namely PBNPs-PEI@AuNCs, was synthesized from PBNPs as the loading carrier, polyethyleneimine (PEI) as the cross-linking agent, and AuNCs directly combined on the surface of PBNPs. The AFB1 Apt was then modified on the PBNPs-PEI@AuNCs to form a PBNPs-PEI@AuNCs-Apt probe, whereby when AFB1 is present, AFB1 is specifically captured by the probe. Meanwhile, the MNPs@antibody was also introduced to capture AFB1, thereby forming a "sandwich" structure compound. After magnetic separation, high temperature was applied to this "sandwich" structure compound to induce the denaturation of the Apt. Then the fluorescent/colorimetric/photothermal signals were collected from the PBNPs-PEI@AuNCs@Apt to give information on its related condition. The detection limits of the biosensor were 0.64 × 10-14, 0.96 × 10-14, and 0.55 × 10-12 g mL-1 for the three signals, which were outputted independently and could be verified with each other to ensure the accuracy of the results. Moreover, the colorimetric and photothermal strategies with this probe do not require large-scale instruments, providing a promising choice for achieving the rapid field detection of AFB1.
Subject(s)
Aflatoxin B1 , Biosensing Techniques , Ferrocyanides , Gold , Metal Nanoparticles , Aflatoxin B1/analysis , Aflatoxin B1/chemistry , Gold/chemistry , Biosensing Techniques/methods , Ferrocyanides/chemistry , Metal Nanoparticles/chemistry , Aptamers, Nucleotide/chemistry , Limit of Detection , Colorimetry/methods , Food Contamination/analysis , Polyethyleneimine/chemistryABSTRACT
Ratiometric fluorescence detection is endowed with higher accuracy than single fluorescence signal assay. In this work, we construct a ratiometric fluorescence probe for the facile quantification of sulfadimethoxine (SDM) in foods. By wrapping N-doped carbon dots (N-CDs) and gold nanoclusters (AuNCs) into zeolitic imidazolate framework-8 (ZIF-8), the nanocomposite of N-CDs/AuNCs@ZIF-8 is facilely prepared and emits two fluorescence including 475 nm from N-CDs and 650 nm from AuNCs. Since bovine serum albumin (BSA) is the stabilizer of AuNCs, SDM can form a complex with BSA, resulting in the fluorescence quenching of AuNCs at 650 nm by a static quenching mechanism. In contrast, SDM has a rare influence on the fluorescence of N-CDs (475 nm). As a result, the use of the probe of N-CDs/AuNCs@ZIF-8 for SDM detection enables simultaneous measurement of response signal and reference signal. Under the optimal condition, the SDM assay based on the probe has a good linear relationship within 10 to 2 × 106 ng/mL and the limit of detection (LOD) is low to 1.064 ng/mL. In addition, the fluorescent probe shows good reliability for the detection of SDM in practical food samples.
ABSTRACT
This work reports an improved enzyme-linked immunosorbent assay (ELISA) via the interaction between prussian blue nanoparticles (PBNPs) and amines for aflatoxin B1 (AFB1) detection. The effect of different amines on the structure and properties of PBNPs was systematically investigated. Amines with pKb < 7, like ethylenediamine (EDA), can decompose structure of PBNPs, leading to the reduction of extinction coefficient and photothermal effect. Whereas, amines with large pKb > 7, such as o-phenylenediamine (OPD), could undergo catalytic oxidation by PBNPs, resulting in the production of fluorescent and colored oxidation products. Accordingly, EDA and OPD were used to construct improved ELISA. Specifically, silica nanoparticles, on which AFB1 aptamer and amino binding agent (ethylenediaminetetraacetic acid disodium salt, EDTAâ¢2Na) were previously assembled via carboxyl-amino linkage, are anchored to microplates by AFB1 and antibody. EDA concentration can be regulated by EDTAâ¢2Na to affect extinction coefficient and photothermal effect of PBNPs, thereby achieving visual colorimetric and portable photothermal signal readout (Model 1). OPD concentration can also be controlled by EDTAâ¢2Na, thus generating colorimetric and ultrasensitive fluorescent signals through PBNPs catalysis (Model 2). The proposed strategy not only opens new avenue for signal readout mode of biosensing, but also provides universal technique for hazards.
Subject(s)
Biosensing Techniques , Ferrocyanides , Nanoparticles , Aflatoxin B1/analysis , Amines , Nanoparticles/chemistry , Enzyme-Linked Immunosorbent Assay , Biosensing Techniques/methods , Limit of DetectionABSTRACT
BACKGROUND: Dichlorvos (DDVP), as a highly effective insecticide, is widely used in agricultural production. However, DDVP residue in foodstuffs adversely affects human health. Conventional instrumental analysis can provide highly sensitive and accurate detection of DDVP, while the need of bulky and expensive equipment limits their application in resource-poor areas and on-site detection. Therefore, the development of easily portable sensing platforms for convenient, rapid and sensitive quantification of DDVP is very essential for ensuring food safety. RESULT: A portable colorimetric sensing platform for rapid and sensitive quantification of DDVP is developed based on nanozyme-participated highly efficient chromogenic catalysis. The Fe-Mn bimetallic oxide (FeMnOx) nanozyme possesses excellently oxidase-like activity and can efficiently catalyze oxidation of 3, 3', 5, 5'-tetramethylbenzidine (TMB) into a blue oxide with a very low Michaelis constant (Km) of 0.0522 mM. The nanozyme-catalyzed chromogenic reaction can be mediated by DDVP via inhibiting the acetylcholinesterase (AChE) activity. Thus, trace DDVP concentration-dependent color evolution is achieved and DDVP can be sensitively detected by spectrophotometry. Furthermore, a smartphone-integrated 3D-printed miniature lightbox is fabricated as the colorimetric signal acquisition and processing device. Based on the FeMnOx nanozyme and smartphone-integrated lightbox system, the portable colorimetric sensing platform of DDVP is obtained and it has a wide linear range from 1 to 3000 ng mL-1 with a low limit of detection (LOD) of 0.267 ng mL-1 for DDVP quantification. SIGNIFICANCE: This represents a new portable colorimetric sensing platform that can perform detection of DDVP in foodstuffs with simplicity, sensitivity, and low cost. The work not only offers an alternative to rapid and sensitive detection of DDVP, but also provides a new insight for the development of advanced sensors by the combination of nanozyme, 3D-printing and information technologies.
Subject(s)
Dichlorvos , Pesticides , Humans , Colorimetry , Oxides , Acetylcholinesterase , Catalysis , Hydrogen PeroxideABSTRACT
To mitigate food spoilage caused by microbial contamination and extend the shelf life of food, antibacterial and eco-friendly biological packaging materials as an alternative to petroleum-based plastics is encouraged. Herein, an innovative and green composite film with triple antibacterial activity has been fabricated by introducing prussian blue nanoparticles (PBNPs) into chitosan (CS)-based films blended with gelatin (Gel) for the preservation of food, named CS/Gel/PB film. Due to the incorporation of PBNPs, CS/Gel/PB film exhibits enhanced mechanical, barrier and water resistance, and thermal abilities. The inherent bacterial trapping and killing capabilities of CS (contact killing), photothermal/photodynamic killing based on the excellent photothermal property of PBNPs under NIR irradiation synergistically facilitate the sterilization against Escherichia coli and Staphylococcus aureus (antibacterial ratio = 99.99 %). The film exhibits outstanding preservation capability in product storage, significantly extending the shelf life of strawberry and pork to 15 and 7 days, respectively. Meanwhile, the cytotoxicity assessment of CS/Gel/PB against HepG2 cells ascertains a cell viability exceeding 96 %, indicating a negligible toxicity level. Additionally, this film also exhibits superior biodegradability (preliminary degradation on the 10th day and completion on the 40th day) compared with PE film. Overall, these properties demonstrate great potential of CS/Gel/PB film as a novel packaging material.
ABSTRACT
Effective identification of sulfur ions (S2-) in foodstuff is crucial for food safety and human health, but it remains challenging. Traditional single-mode colorimetric sensing methods are simple and sensitive, but are prone to interference from colored substances which can lead to false positives or negatives results. Herein, we develop a novel "mix-response" biosensor for colorimetric and photothermal dual-mode detection of S2- with good simplicity, sensitivity and portability. In this biosensor, silver-doping Prussian blue nanoparticle (SPB NPs) was used as signal output component, which not only exhibits blue color characteristics, but also has photothermal conversion properties activated by near-infrared (NIR) laser. Upon increasing the S2- concentration, the prepared SPB NPs undergo etching, leading to the formation of new silver sulfide precipitation (Ag2S), along with different colorimetric and photothermal response signals. For the portable visualization of S2-, the color information was recorded by a smartphone in combination with RGB (red channel) analysis and the evolution of the photothermal signal was documented by a thermal imager. The introduction of smartphone and handheld thermal imager in this "mix-response" biosensor makes it suitable for on-site quantitative detection of S2- without sophisticated instrument. Moreover, the development of this "mix-response" biosensor does not need the use of recognition probes (e.g. aptamers and reaction intermediates), thereby simplifying the construct procedures of sensing strategies and improving the economic efficiency of detection. More importantly, the photothermal response signals can overcome the interference of colored substances in foods, thereby reducing the false positives or negatives of the detection results.
ABSTRACT
Core-shell quantum dots suffer from photobleaching by light at wavelengths longer than their emission wavelengths. That is, QD photobleaching can be triggered by photons with low energies that are insufficient to pump electrons into the conduction band. The most probable reason is that electrons are pumped into a surface state and then nonradiatively decayed as in conventional photobleaching.
Subject(s)
Light , Photobleaching , Quantum Dots , Models, ChemicalABSTRACT
As a common raw material of industrial products, bisphenol A (BPA) is widely used in the production of food contact materials, and there is a high risk of exposure in food. However, BPA is a well-known endocrine disruptor and poses a serious threat to human health. Herein, a fluorescent sensing platform of BPA based on enzymatic oxidation-mediated fluorescence quenching of silicon nanoparticles (SiNPs) is established and used to the detection of BPA in food species. The SiNPs are prepared with a facile one-step synthesis and emit bright green fluorescence. BPA can be oxidized by horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) to form a product which can quench the fluorescence of SiNPs through electron transfer. There is a good linear relationship between the fluorescence intensity and BPA concentration in the range of 1-100 µM. Therefore, a fluorometry of BPA is established with a low limit of detection (LOD) of 0.69 µM. This method has been applied to the determination of BPA in mineral drinking water, orange juice, and milk with satisfactory results. The fluorescent sensor of BPA based on SiNPs has favorable application foreground in the field of food safety analysis.
Subject(s)
Nanoparticles , Silicon , Humans , Spectrometry, Fluorescence/methods , Hydrogen Peroxide/analysis , Fluorescent DyesABSTRACT
The key factor of realizing super-resolution optical microscopy at the single-molecule level is to separately position two adjacent molecules. An opportunity to independently localize target molecules is provided by the intermittency (blinking) in fluorescence of a quantum dot (QD) under the condition that the blinking of each emitter can be recorded and identified. Herein we develop a spectral imaging based color nanoscopy which is capable of determining which QD is blinking in the multicolor QD complex through tracking the first-order spectrum, and thus, the distance at tens of nanometers between two QDs is measured. Three complementary oligonucleotides with lengths of 15, 30, and 45 bp are constructed as calibration rulers. QD585 and QD655 are each linked at one end. The measured average distances are in good agreement with the calculated lengths with a precision of 6 nm, and the intracellular dual-color QDs within a diffraction-limited spot are distinguished.
Subject(s)
Microscopy , Quantum Dots , Cell Line , Color , Humans , Oligonucleotides/chemistryABSTRACT
In this paper, a portable fluorescence-based functional hydrogel loaded with ß-d-glucose pentaacetate (ß-D-GP) is designed for high-sensitive quantification of amine vapor and visual monitoring of freshness of shrimp. We found for the first time that amine vapor can mediate ß-D-GP to generate photoluminescent polymer-carbon nanodots (PCNDs) with good optical properties. On this basis, a functional hydrogel sensing platform is simply formed by solidifying ß-D-GP in agarose hydrogels. When exposure to the volatile amines released from the spoilage of shrimp, ß-D-GP in hydrogel is immediately mediated by amines to generate PCNDs, resulting in obvious fluorescence-based color variation of functional hydrogel. Notably, a smartphone is used to obtain digital photographs and RGB (Red/Green/Blue) information of hydrogels for on-site quantitative analysis. The gray value of G/(R + B) of hydrogel shows good linearity with trimethylamine (TMA) vapor concentration in the range of 0-59.49 × 10-9 mol dm-3. More importantly, the G/(R + B) value of functional hydrogel is successfully used to assess the freshness of shrimp. Consequently, this strategy provides a low-cost, portable fluorescence analysis device with promising applications in achieving high-sensitive, nondestructive, and on-site food safety evaluation of animal-derived aquatic products.
Subject(s)
Amines , Carbon , Animals , Glucose/analogs & derivatives , Hydrogels , PolymersABSTRACT
Dual-mode sensing with a two-signal read-out is conducive to the improvement of detection accuracy. Herein, a fluorescent and scattering dual-mode chemosensor for tetracycline (TC) is proposed based on a carbon dot@cerium-guanosine monophosphate (CD@GMP-Ce) coordination polymer network. The inexpensive CD@GMP-Ce was prepared by exploiting the adaptive inclusion capability of coordination polymers and possessed remarkable fluorescence and strong Rayleigh scattering. The functional CD@GMP-Ce demonstrated fluorescence and scattering, the two optical-signal responses to TC simultaneously. Based on TC-specific fluorescence and scattering decline, the dual-mode detection of TC was established and the probe's detection limits were 43 nM in the fluorescence mode and 77 nM in the scattering mode, respectively. Furthermore, the potential application of the dual-mode sensor was verified by measuring TC in milk and tap-water samples. The study not only provides a new perspective for the development of assay methods for TC but also expands the applications of cerium coordination polymers.
Subject(s)
Cerium , Polymers , Guanosine Monophosphate , Carbon , Spectrometry, Fluorescence/methods , Tetracycline , Anti-Bacterial Agents , Fluorescent DyesABSTRACT
As a gold standard technique, enzyme-linked immunosorbent assay (ELISA) organically combines immunoreactions between antigens and antibodies with enzyme catalysis. The use of ELISA has contributed to advances in applications such as clinical diagnosis, food quality control, and environmental monitoring. However, conventional ELISA suffer from the moderate sensitivity and reliance on enzyme activity, which make it impossible to reliably and inexpensively detect trace targets. The nanotechnology boom has yielded exciting developments in designing nanomaterial-based improved ELISA in recent years. In this review, we attempt to comprehensively describe the improvements in ELISA methodology based on nanomaterials, with a focus on the mode of signal detection, such as colorimetric, fluorescent, electrochemical, photothermal, and Raman scattering sensing. We particularly emphasized on how nanomaterials are used as loading carriers, enzyme mimics, and signal reporters. This review concerns on partially representative examples and describes novel concepts and promising applications, rather than being exhaustive. Finally, we outline the challenges and perspectives, hopefully provide brief guideline to develop neotype improved ELISA.
Subject(s)
Nanostructures , Catalysis , Colorimetry , Enzyme-Linked Immunosorbent Assay , NanotechnologyABSTRACT
This work aims to develop a novel multimode (photothermal/colorimetric/fluorescent) nanozyme-linked immunosorbent assay (NLISA) based on the in situ generation of Prussian blue nanoparticles (PBNPs) on the surface of magnetic nanoparticles (MNPs). Being considered the most toxic among the mycotoxins, aflatoxin B1 (AFB1) was chosen as the proof-of-concept target. In this strategy, MNPs, on which an AFB1 aptamer was previously assembled via streptavidin-biotin linkage, are anchored to 96-well plates by AFB1 and antibody. In the presence of HCl and K4Fe(CN)6, PBNPs formed in situ on the MNP surface, thereby achieving photothermal and colorimetric signal readout due to their photothermal effect and intrinsic peroxidase-like activity. Based on fluorescence quenching by MNPs, Cy5 fluorescence was recovered by the in situ generation of PBNPs to facilitate ultrasensitive fluorescence detection. Photothermal and colorimetric signals allow portable/visual point-of-care testing, and fluorescent signals enable accurate determination with a detection limit of 0.54 fg/mL, which is 6333 and 28 times lower than those of photothermal and colorimetric analyses, respectively. We expect that this proposed multimode NLISA can not only reduce the false-positive/negative rates through the multisignal crossdetection in AFB1 monitoring but also provide a universal way of sophisticated instrumentation-free, easy-to-use, cost-effective, and highly sensitive detection of other food hazards.
Subject(s)
Aflatoxin B1/analysis , Biosensing Techniques/methods , Ferrocyanides/chemistry , Food Contamination/analysis , Immunosorbents/chemistry , Nanoparticles/chemistry , Acetic Acid/analysis , Arachis/chemistry , Biological Assay , Wine/analysisABSTRACT
Quantum dots (QDs) are promising fluorescent tags for microarrays. Because most microarrays are analyzed under dry conditions, it is necessary to examine the photo properties of QDs in air. We demonstrate that the photophysical characteristics of individual quantum dots are different at the liquid/solid interface compared with QDs at the air/solid interface by observing them through a wide-field fluorescence microscope. QDs in air show higher photo-stability, higher fluorescence signal, slower spectral blue shift rate, less blinking and shorter bulk fluorescence lifetime than those in solution. These beneficial properties indicate QDs are good alternative fluorescent probes for microarrays.
Subject(s)
Physics , Quantum Dots , Air , Fluorescent Dyes , Microscopy, FluorescenceABSTRACT
⢠Smart usage of single molecule photobleaching technology and dual-color fluorescence colocalization is of critical importance for exploiting the sensing platform. Here, we provide the detailed protocols related to the article "A split aptamer sensing platform for highly sensitive detection of theophylline based on dual-color fluorescence colocalization and single molecule photobleaching" (published online by Biosensors and Bioelectronics) (Liu et al., 2020). The protocols contain: (1) how to clean the slides; (2) how to prepare the probe and detection sample; (3) Single molecule imaging; 4) Data processing by using the Image J. Finally, we used a simple model to confirm the feasibility of the method for integrating dual-color fluorescence colocalization and single molecule photobleaching technology on the theophylline sensing platform. ⢠A simple, ultrasensitive method for the detection of theophylline. ⢠The method is easily comprehensible. ⢠Both strategy formulation and data processing are simple, learnability, and highly reproducible.
ABSTRACT
A new split aptamer sensing platform is developed for highly sensitive and selective detection of theophylline based on single molecule photobleaching (SMPB) technique. The sensing system contains two probes. One is formed by one streptavidin and four biotinylated RNA fragments labelled with fluorescein isothiocyanate (FITC). Each biotinylated RNA fragment contains two repeating aptamer fragments. The other probe is the complementary aptamer fragment labelled with Cy5 dye. The existence of theophylline can trigger the first probe to bind as many as eight Cy5-labelled probes. The average combined number depends on the theophylline concentration and can be measured by SMPB technique. In the sensing system, the dual-color fluorescence colocalization is performed by the red fluorophore (Cy5) and green fluorophore (FITC), in which the red fluorophore is utilized for quantitative counting of photobleaching steps, while the green fluorophore serves as a counting reference to increase detection efficiency. On basis of the principle, an ultra-sensitive sensing platform of theophylline is created with a low limit of detection (LOD) of 0.092 nM. This work provides not only a highly sensitive method for theophylline detection but also a novel perspective for the applications of SMPB technology to construct biosensors.