Sensitive electrical detection of human prion proteins using field effect transistor biosensor with dual-ligand binding amplification.

Field effect transistor (FET) biosensor was considered as a valuable tool for detection of chemical and biological molecules over the past decades [1]. The basic principle of FET biosensor is by converting the variation in the surface potential of gate insulator into the conductivity change in the semiconductor channel. Such kind of direct detection can interestingly offers a simple, label-free and reliable sensing mechanism. However, to configure highly sensitive sensor, the FET signal is strongly depend on the electric field resulting from the binding of charged molecule to the gate surface. Hence, the charge of target molecule is a key factor for electrically-based sensing in which the FET biosensor in some cases hampers to be applied for the detection of low charge density of molecules and neutral molecules. In this research, we designed two approaches of charge amplification including (i) addition of metal ions as second ligand, and (ii) chemical modification via coupling reaction as illustrated at Figure 1. These strategies were performed into the target protein that can increase the charge of proteins, leading to the enhancement of FET signal. Here, we used and detected prion proteins as a model of the target protein. Highly sensitive method for detecting prion proteins is a top priority for early diagnosis and minimizing the spread of the diseases [2], and the amplified FET signal is of utmost importance to meet the goal for improving the sensitivity of prion detection to the lowest concentration as possible. Firstly, by utilizing thiamine as probe molecule, the surface of FET biosensor can specifically interact with the prion protein as confirmed by FET response, atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) analysis [3]. Then, the sensitivity of thiamine-immobilized FET to the prion protein in human serum sample before the use of amplification step was quantitatively determined and achieved at the concentration of 400 pM, which is also lower than the cut-off value (2 nM). Furthermore, the amplification process was applied. As for approach (i), by taking the advantage of specific interaction between prion protein and copper (II) ions (Cu2+), a model of dual-ligand binding consist of thiamine immobilized surface, prion protein and Cu2+was performed and successfully generated an enhancement of FET response. As for approach (ii), we demonstrated the feasibility of protein modification with conventional coupling reaction, converting the carboxylate group (anionic) to the amino group (cationic) of prion protein. These strategies can result higher electrical signals in prion detection based on FET biosensor. The detail of experimental procedure and mechanism will be discussed further in the conference. In summary, the FET biosensor device could provide a promising technique and a pave way for highly sensitive detection of prion protein.

The dosage of 5-fluorouracil (5-FU), which is a widely used for cancer medication, is determined based on body surface area, although efficacy largely depends on the liver function of the individual, resulting that only 21% patients are given an optimal dose of 5-FU in these years. The measurement of the 5-FU concentration in the blood enables us to adjust individual dose adjustment. Conventional methods for 5-FU detection such as an enzyme-linked immunosorbent assay (ELISA) and liquid chromatography are not very suitable for clinical applications because they need time-consuming procedure with expensive equipments. Detection of 5-FU using a field effect transistor (FET) biosensor, which enables rapid and simple measurement, is expected to solve such problems. However, FET biosensor, which detects changes in its surface density due to the adsorption of charged molecules, was unable to detect uncharged 5-FU. In this study, a method for the FET biosensing to detect 5-FU exploiting sequential adsorption of 5-FU modified bovine serum albumin (BSA/5-FU) was proposed. By using this method, FET responses caused by the adsorption of negatively charged BSA/5-FU depending on the 5-FU concentration were detected. The SiO2 surface of the FET gate insulator was exposed to O2 plasma to introduce hydroxyl groups. After the exposure, the surface was exposed to 3-aminopropyltriethoixysilane (APTES), followed by the modification of the cross-linker, glutaraldehyde (GA). A single chain variable fragments (ScFv) and antigen binding fragments (Fab) were allowed to react with each activated GA-modified FET. After the immobilization, the residual aldehyde-groups were treated by ethanolamine to suppress the non-specific adsorption. V g-I d characteristics were measured before and after dripping of both 5-FU and BSA/5-FU on the ScFv- and Fab-immobilized FET biosensors. Finally, threshold voltage shifts (∆V g) caused by the adsorption of BSA/5-FU were obtained. To compare the capture capability of ScFv and Fab, the electrical responses of the FET biosensors functionalized with the two receptors due to the adsorption of BSA/5-FU were measured. The responses of ScFv- and Fab-immobilized FET biosensor caused by dripping of 25 μg/mL BSA/5-FU were +25 mV and +40 mV, respectively. The difference between ΔV g values for these two FET sensors using ScFv or Fab can be ascribed to the difference of affinity [1]. To verify the specificity of Fab-immobilized FET biosensor, ∆V g was measured when human serum albumin (HSA) was dropped on the FET biosensor, and the ∆V g was hardly observed. Additionally, the atomic force microscopic (AFM) images on the FET gate surface shows that the size of observed particles matches the size of BSA/5-FU, while the surface morphology and roughness are not significantly changed. These results indicated that Fab-immobilized surface specifically captured BSA/5-FU. To investigate the quantitative detectability of the Fab-immobilized FET biosensor, we measured the FET responses corresponding to the amount of adsorbed BSA/5-FU, which was related with the concentrations of 5-FU. As a result, the magnitude of ∆V g by dripping of 1000 ng/mL 5-FU and 25 μg/mL BSA/5-FU was reduced to +12 mV compared with the response of 25 µg/mL BSA/5-FU (Figure 1). These results can be attributed to that the adsorbed 5-FU inhibited the adsorption of BSA/5-FU to the Fab-immobilized surface. Therefore, we conclude that the detection of 5-FU using the FET biosensors by applying the charged BSA/5-FU is a promising simple method for monitoring the concentration of 5-FU.Reference:[1] Y. Reiter, et al., J. Biol. Chem., 269, 28, 18327-18331 (1994).Figure 1 V g-I d characteristics of Fab-immobilized FET biosensor before and after dripping of (a) 0 ng/mL 5-FU and 25 µg/mL BSA/5-FU or (b) 1000 ng/mL 5-FU and 25 µg/mL BSA/5-FU. Figure 1

Signal amplification in electrochemical detection of buckwheat allergenic protein using field effect transistor biosensor by introduction of anionic surfactant

The highly sensitive and cost-free field effect transistor (FET) biosensor for specific detection of DNA and streptavidin based on Zinc Oxide Nanowires (NWs) to develop the next generation biosensors in label-free approach under electrochemical gate modulation has been materialized. The electronic properties of NWs were changed by binding of charged analytes to NWs surface under electrochemical gate modulation. Due to highly chemically inertness, immobilization of the target analyte receptors on the NWs could be conducted through electrochemical treatment. For materialization of DNA binding, gold nanoparticles (Au NPs) were used to functionalize ZnO NWs surface. In this work, a liquid gated FET based on NWs was configured to detect the complementary DNA strand hybridization as well as the protein streptavidin at very low concentration. The biosensor could reach to sensitivity of attomolar. The sensitivity of response was check and biosensor showed the sensitivity ranges from 10−18 to 10−12 mol.L−1. The FET biosensor could distinguish a complementary strand from a single nucleotide polymorphism (SNP) containing strand. The limit of detection (LOD) reached to15 aM. The NPs decoration on the NWs surface brought about the high detection limit. The NPs decoration was being a promising method and scientific approach for biomolecules detection.

Proteins have been one of the most important biomarkers for diagnosing diseases, and field-effect transistor (FET) biosensors possess high sensitivity; are label-free; and feature real-time detection, rapidity, and easy integration for protein detection. FET biosensors are mainly made up of FET parts, such as channel materials, and bio parts, such as receptors. This Tutorial provides an in-depth exploration of FET biosensors for protein detection from the composition perspective and discusses the commercialization of point-of-care diagnostics of proteins based on FET biosensors.

<p>The efficient detection of disease-relevant biomolecules in untreated clinical samples is highly desired, especially for acute diseases. Field-effect transistor (FET) biosensors allow label-free and rapid detection of biomolecules through the measurement of their intrinsic charges. However, the sensitivity of FET biosensors would be undermined by the charge screening effect in practical biological media with high ionic strength. Here, we report the design and performance of a nanostructured interface-engineered field effect transistor (NIE FET) biosensor for highly sensitive detection of cardiovascular disease (CVD)-associated miRNAs in serum samples. Molecular dynamic simulations and electrochemical characterizations demonstrate that the nanostructured interface with concave regions alleviates the charge screening effect and enlarges the Debye length. The rationally designed NIE FET biosensor exhibits high sensitivity and reproducibility in detecting miRNA in untreated serum samples with a detection limit of pM level. Benefiting from its excellent detection capabilities, NIE FET reveals the relationship between miRNAs and CVDs and realizes the effective classification of different CVD types with the help of machine learning algorithms. The construction of NIE FET defines a robust strategy for electrical biomolecular detection in practical clinical samples.</p>

Field-effect transistor (FET) biosensors based on two-dimensional (2D) materials have drawn significant attention due to their outstanding sensitivity. However, the Boltzmann distribution of electrons imposes a physical limit on the subthreshold swing (SS), and a 2D-material biosensor with sub-60 mV/dec SS has not been realized, which hinders further increase of the sensitivity of 2D-material FET biosensors. Here, we report tunnel FETs (TFETs) based on a SnSe2/WSe2 heterostructure and observe the tunneling effect of a 2D material in aqueous solution for the first time with an ultralow SS of 29 mV/dec. A bilayer dielectric (Al2O3/HfO2) and graphene contacts, which significantly reduce the leakage current in solution and contact resistance, respectively, are crucial to the realization of the tunneling effect in solution. Then, we propose a novel biosensing method by using tunneling current as the sensing signal. The TFETs show an extremely high pH sensitivity of 895/pH due to ultralow SS, surpassing the sensitivity of FET biosensors based on a single 2D material (WSe2) by 8-fold. Specific detection of glucose is realized, and the biosensors show a superb sensitivity (3158 A/A for 5 mM), wide sensing range (from 10-9 to 10-3 M), low detection limit (10-9 M), and rapid response rate (11 s). The sensors also exhibit the ability of monitoring glucose in complex biofluid (sweat). This work provides a platform for ultrasensitive biosensing. The discovery of the tunneling effect of 2D materials in aqueous solution may stimulate further fundamental research and potential applications.

Viral infections are one of the main causes of deaths and economic losses around the globe, and effective virus detection methods are essential for epidemic prevention and control. Most existing detection methods have problems such as high false negative/positive rates, slow responses, high costs, and dependence on professional equipment and personnel, which are not conducive to the rapid and accurate detection of viruses. Field effect transistor (FET) biosensors have attracted widespread attention due to their advantages of label-free detection, high sensitivity, fast responses, real-time measurement, low power consumption, and small sizes for portability. This article first briefly describes the basic situation of viruses and the structure and detection principle of FET biosensors. Subsequently, it delves into the research achievements in the application of FET biosensors in the detection of influenza viruses, hepatitis viruses, human immunodeficiency virus, and severe acute respiratory syndrome coronavirus 2. Finally, we make a comprehensive summary and reasonable outlook on the role played by FET biosensors in biomedicine.

Effect of human serum on the electrical detection of amyloid-β fibrils in biological environments using azo-dye immobilized field effect transistor (FET) biosensor

Low-dimensional semiconductor-based field-effect transistor (FET) biosensors are promising for label-free detection of biotargets while facing challenges in mass fabrication of devices and reliable reading of small signals. Here, we construct a reliable technology for mass production of semiconducting carbon nanotube (CNT) film and FET biosensors. High-uniformity randomly oriented CNT films were prepared through an improved immersion coating technique, and then, CNT FETs were fabricated with coefficient of performance variations within 6% on 4-in. wafers (within 9% interwafer) based on an industrial standard-level process. The CNT FET-based ion sensors demonstrated threshold voltage standard deviations within 5.1 mV at each ion concentration, enabling direct reading of the concentration information based on the drain current. By integrating bioprobes, we achieved detection of biosignals as low as 100 aM through a plug-and-play portable detection system. The reliable technology will contribute to commercial applications of CNT FET biosensors, especially in point-of-care tests.

Electrical detection of RNAs using transistor-based biosensors has attracted attention as a strategy for medical diagnosis and environmental monitoring. Herein, we demonstrated a proof-of-concept for specific, sensitive, and label-free RNA detection using a field-effect transistor (FET) biosensor with signal amplification by ternary initiation complexes (SATIC), which is an isothermal one-step nucleic acid amplification initiated by the combination of target RNA, circular DNA template and DNA primer. The SATIC system-applied FET biosensor specifically and quantitatively detected the target RNA with a single-nucleotide difference via the negative charges derived from the amplification products formed by a nucleic acid amplification reaction with φ29 DNA polymerase on the gate surface. In particular, the control of the amplification time allowed the detection of target RNA molecules over a wide concentration range, resulting in a detection limit of up to 6 copies/μL. Therefore, a transistor-based bioassay using the SATIC system could be useful for simple and sensitive nucleic acid analysis.

A pair of single chain Fv fragment (scFv) fusion proteins were constructed and characterized. Antibody chips using the pair were designed for sensitive detection of prion protein. Phage displayed antibody library was synthesized by immunizing mice with thioredoxin‐mature bovine prion fusion protein (TrxA‐bPrPc). After five rounds of panning against recombinant bovine prion protein (rb‐PrPc) and ELISA test, two positive clones with high affinity to rb‐PrPc, named Z163 and Z186, were obtained. They were conjugated with a linker‐streptavidin binding protein (SBP) or human IgG1 constant fragment (Fc) to form the scFv fusion protein pair Z186‐L‐SBP/Z163‐Fc. Western blot experiments showed that the scFv fusion pair specifically interacted with the line epitopes of the protease resistant core region bPrP27‐30. Surface plasmon resonance (SPR) sensorgrams revealed that the equilibrium dissociation constants of the interactions with rb‐PrPc were 3.24×10−8 M, 8.82×10−8M, and 8.10×10−9 M for Z186‐L‐SBP, Z163, and Z163‐Fc, respectively. All binding reactions followed rapid association and slow dissociation kinetics. As a detection pair, Z186‐L‐SBP functioned as a capture probe and was immobilized on the streptavidin coated slides to form reactive layer of the antibody chip, and Z163‐Fc labeled with fluorescence dye Cy3 functioned as a detection probe generating fluorescence signal. The antibody chip could detect existence of rb‐PrPc with detection limit of 1 pg/ml.

A high sensitivity field effect transistor biosensor for methylene blue detection utilize graphene oxide nanoribbon

Transition-metal dichalcogenides (TMDs), including molybdenum disulfide (MoS2) and tungsten disulfide (WS2), with appealing properties have recently become promising alternatives to graphene with semimetal and low on/off current ratio properties as the sensing channel in field-effect transistor (FET) biosensors. However, the efficiency of DNA-based FET devices strongly depends on how DNA probes are tethered to the nanomaterial channels. As against covalent attachment, simple DNA physisorption has become increasingly popular, and a DNA sequence with strong affinity for nanomaterials is still highly sought after. Recently, poly-cytosine (poly-C) DNA was found to be strongly adsorbed to many common nanomaterials, including WS2. Herein, a diblock DNA probe containing a (poly-C) (C15) was used to attach to a chemical vapor deposition (CVD)-grown monolayer WS2 surface; meanwhile, the target complementary DNA (cDNA) was hybridized to the other block of the DNA probe. The biosensor developed following this strategy led to a limit of detection down to 3 aM within a concentration range spanning over approximately 7 orders of magnitude (10–16 to 10–9 M), which was lower than those of the previously reported TMDs and a good competitor to graphene FET DNA biosensors. Moreover, the proposed WS2 FET DNA biosensor showed high specificity capable of distinguishing the cDNA from non-cDNA, one-base mismatched DNA, two-base mismatched DNA, and three-base mismatched DNA, making our strategy an exciting avenue for disease diagnosis. The authors are convinced that this work extends the CVD synthesis of WS2 and its promise in biosensing application-based FETs.

We describe the fabrication of highly sensitive graphene based field effect transistor (FET) biosensors with a cost-effective approach and their application in label-free Deoxyribonucleic acid (DNA) detection. Chemical vapor deposition (CVD) grown graphene layers were used to achieve mass production of FET devices via conventional photolithographic patterning. Non-covalent functionalization of the graphene layer with 1-Pyrenebutanoic acid succinimidyl ester ensures high conductivity and sensitivity of the FET device. The present device could reach a detection limit as low as 3 x 10(-9) M.