Potential diagnostic applications of biosensors: current and future directions

This review describes recent advances in biosensors of potential clinical applications. Biosensors are becoming increasingly important and practical tools in pathogen detection, molecular diagnostics, environmental monitoring, food safety control as well as in homeland defense. Electrochemical biosensors are particularly promising toward these goals arising due to several combined advantages including low-cost, operation convenience, and miniaturized devices. We review the clinical applications of electrochemical biosensors based on a few selected examples, including enzyme-based biosensors, immunological biosensors and DNA biosensors.     

The Use of Whole-Cell Biosensors to Detect and Quantify Compounds or Conditions Affecting Biological Systems

A new and promising technique in microbial ecology and environmental biology is the use of whole-cell bacterial biosensors. This minireview describes the use of such biosensors for detection and quantification of various compounds and other conditions affecting bacterial expression of different genes. Three types of biosensors (nonspecific, stress-induced, and specific biosensors) are described including their use in different environments. We present tables of published biosensors, including gene fusions, host organisms, and environments in which they are used. We here describe the use of different reporter genes in the construction of biosensors and discuss their use as tools for monitoring the bioavailability of pollutants and their potential use in studying microbial ecology in general.     

Covalent enzyme immobilization by poly(ethylene glycol) diglycidyl ether (PEGDE) for microelectrode biosensor preparation

Poly(ethylene glycol) diglycidyl ether (PEGDE) is widely used as an additive for cross-linking polymers bearing amine, hydroxyl, or carboxyl groups. However, the idea of using PEGDE alone for immobilizing proteins on biosensors has never been thoroughly explored. We report the successful fabrication of microelectrode biosensors based on glucose oxidase, d-amino acid oxidase, and glutamate oxidase immobilized using PEGDE. We found that biosensors made with PEGDE exhibited high sensitivity and a response time on the order of seconds, which is sufficient for observing biological processes in vivo. The enzymatic activity on these biosensors was highly stable over several months when they were stored at 4 °C, and over at least 3d at 37 °C. Glucose microelectrode biosensors implanted in the central nervous system of anesthetized rats reliably monitored changes in brain glucose levels induced by sequential administration of insulin and glucose. PEGDE provides a simple, low cost, non-toxic alternative for the preparation of in vivo microelectrode biosensors.     

Development of renewable surface biosensors to meet industrial needs for measurement of glucose in fruit juices.

Most biosensors reported to date have been prepared, studied and used under laboratory conditions. The feasibility of a very great number of biosensors seems to be demonstrated and their characteristics, very often, established as corresponding to the demands of the modern analysis. The operational stability of the biosensors, according to authors, is almost always acceptable. The long term storage, with analytical quality conservation that is necessary to commercialise products, has rarely been studied. The stability of biosensors has to remain not only during the fabrication step or their subsequent utilisation, but also throughout the whole commercial shelf-life of the sensor, from producer to end user, through wholesaler and/or retailer. We developed the manufacturing processes, on a large scale, of renewable surface electrodes modified with enzymes such as oxidoreductases. The process consisting of several steps is described and the analytical behaviours of resulting biosensors is studied and correlated with the effects of different constraints applied during the fabrication process.     

Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors

Fluorescence resonance energy transfer (FRET)-based molecular biosensors serve as important tools for studying protein activity in live cells and have been widely used for this purpose over the past decade. However, FRET biosensors are rarely used in the context of the live organism because of the inherent high cellular complexity and imaging challenges associated with the three-dimensional environment. Here we provide a protocol for using single-chain intramolecular FRET-based biosensors in early development. We provide a general protocol for FRET ratio imaging in embryos, including the data-acquisition conditions and the algorithm for ratio image generation. We then use the pRaichu RacFRET biosensor to exemplify the adaptation and optimization of a particular biosensor for use in live zebrafish embryos. Once an optimized biosensor is available, the complete procedure, including introduction of the probes into embryos, imaging and data analysis, requires 2-3 d.     

Progress of new label-free techniques for biosensors: a review

The detection techniques used in biosensors can be broadly classified into label-based and label-free. Label-based detection relies on the specific properties of labels for detecting a particular target. In contrast, label-free detection is suitable for the target molecules that are not labeled or the screening of analytes which are not easy to tag. Also, more types of label-free biosensors have emerged with developments in biotechnology. The latest developed techniques in label-free biosensors, such as field-effect transistors-based biosensors including carbon nanotube field-effect transistor biosensors, graphene field-effect transistor biosensors and silicon nanowire field-effect transistor biosensors, magnetoelastic biosensors, optical-based biosensors, surface stress-based biosensors and other type of biosensors based on the nanotechnology are discussed. The sensing principles, configurations, sensing performance, applications, advantages and restriction of different label-free based biosensors are considered and discussed in this review. Most concepts included in this survey could certainly be applied to the development of this kind of biosensor in the future.     

Exposing culprit organic pollutants: a review

There is a continuing need for monitoring the health of the environment due to the presence of pollutants. Here, we review the development and attributes of biosensors by which bacteria have been genetically modified to express the luminescence genes, i.e. to glow, in a quantified manner, in response to pollutants. We have concentrated on the detection of organic hydrocarbon pollutants and discussed the molecular mechanisms by which some of these chemicals act as effector molecules on the respective regulatory systems. The future of environmental biosensors is predictably bright. As more knowledge is gathered on the sensing regulatory component, the possibility of developing targeted or pollutant-specific biosensors is promising. Moreover, the repertoire of biosensors for culprit organic pollutants is expected to be enlarged through advances in genomics technology and identification of new sensory or receptor molecules. The need for pollutant detection at concentrations in the parts per trillion range or biosensors configured in a nanoscale is anticipated.     

Insight of smart biosensors for COVID-19: A review

The review discusses the diagnostic application of biosensors as point-of-care devices in the COVID-19 pandemic. Biosensors are important analytical tools that can be used for the robust and effective detection of infectious diseases in real-time. In this current scenario, the utilization of smart, efficient biosensors for COVID-19 detection is increasing and we have included a few smart biosensors such as smart and intelligent based biosensors, plasmonic biosensors, field effect transistor (FET) biosensors, smart optical biosensors, surface enhanced Raman scattering (SERS) biosensor, screen printed electrode (SPE)-based biosensor, molecular imprinted polymer (MIP)-based biosensor, MXene-based biosensor and metal–organic frame smart sensor. Their significance as well as the benefits and drawbacks of each kind of smart sensor are mentioned in depth. Furthermore, we have compiled a list of various biosensors which have been developed across the globe for COVID-19 and have shown promise as commercial detection devices. Significant challenges in the development of effective diagnostic methods are discussed and recommendations have been made for better diagnostic outcomes to manage the ongoing pandemic effectively.     

Microfluidic-integrated biosensors: Prospects for point-of-care diagnostics

There is a growing demand to integrate biosensors with microfluidics to provide miniaturized platforms with many favorable properties, such as reduced sample volume, decreased processing time, low cost analysis and low reagent consumption. These microfluidics-integrated biosensors would also have numerous advantages such as laminar flow, minimal handling of hazardous materials, multiple sample detection in parallel, portability and versatility in design. Microfluidics involves the science and technology of manipulation of fluids at the micro- to nano-liter level. It is predicted that combining biosensors with microfluidic chips will yield enhanced analytical capability, and widen the possibilities for applications in clinical diagnostics. The recent developments in microfluidics have helped researchers working in industries and educational institutes to adopt some of these platforms for point-of-care (POC) diagnostics. This review focuses on the latest advancements in the fields of microfluidic biosensing technologies, and on the challenges and possible solutions for translation of this technology for POC diagnostic applications. We also discuss the fabrication techniques required for developing microfluidic-integrated biosensors, recently reported biomarkers, and the prospects of POC diagnostics in the medical industry.     

Sensitivity Analysis of Gold Nanorod Biosensors for Single Molecule Detection

Single protein molecule detection is important for investigating molecular behavior and diagnosing diseases at an early stage. Gold nanorod (GNR) biosensors have shown promise for label-free detection of single protein molecules. However, for widespread applications of GNR biosensors with high sensitivity, detail studies are needed to understand the effects of the sensing environment and the molecular binding dynamics on the sensitivity. In this work, a comprehensive theoretical analysis with variable substrate, buffer, ligand, and binding position of the target molecules shows that GNR biosensors are highly sensitive for single molecule detection of biological samples including critical pathogens such as cancer marker thyroglobulin and human immunodeficiency virus (HIV) marker glycoprotein. We also propose and show that a GNR biosensor with a dielectric cladding layer on the body increases the sensitivity by orders of magnitude compared to other state-of-the-art biosensors.

     

Slow light engineering for high Q high sensitivity photonic crystal microcavity biosensors in silicon

Current trends in photonic crystal microcavity biosensors in silicon-on-insulator (SOI), that focus on small and smaller sensors have faced a bottleneck trying to balance two contradictory requirements of resonance quality factor and sensitivity. By simultaneous control of the radiation loss and optical mode volumes, we show that both requirements can be satisfied simultaneously. Microcavity sensors are designed in which resonances show highest Q～9300 in the bio-ambient phosphate buffered saline (PBS) as well as highest sensitivity among photonic crystal biosensors. We experimentally demonstrated mass sensitivity 8.8atto-grams with sensitivity per unit area of 0.8pg/mm(2). Highest sensitivity, irrespective of the dissociation constant K(d), is demonstrated among all existing label-free optical biosensors in silicon at the concentration of 0.1μg/ml.     

Advances in Cell‐Free Biosensors: Principle,Mechanism, and Applications

Synthetic biology has promoted the development of biosensors as tools for detecting trace substances. In the past, biosensors based on synthetic biology have been designed on living cells, but the development of cell biosensors has been greatly limited by defects such as genetically modified organism problem and the obstruction of cell membrane. However, the advent of cell‐free synthetic biology addresses these limitations. Biosensors based on the cell‐free protein synthesis system have the advantages of higher safety, higher sensitivity, and faster response time over cell biosensors, which make cell‐free biosensors have a broader application prospect. This review summarizes the workflow of various cell‐free biosensors, including the identification of analytes and signal output. The detection range of cell‐free biosensors is greatly enlarged by different recognition mechanisms and output methods. In addition, the review also discusses the applications of cell‐free biosensors in environmental monitoring and health diagnosis, as well as existing deficiencies and aspects that should be improved. In the future, through continuous improvement and optimization, the potential of cell‐free biosensors will be stimulated, and their application fields will be expanded.     

An update on genetically encoded lipid biosensors

Specific lipid species play central roles in cell biology. Their presence or enrichment in individual membranes can control properties or direct protein localization and/or activity. Therefore, probes to detect and observe these lipids in intact cells are essential tools in the cell biologist’s freezer box. Herein, we discuss genetically encoded lipid biosensors, which can be expressed as fluorescent protein fusions to track lipids in living cells. We provide a state-of-the-art list of the most widely available and reliable biosensors and highlight new probes (circa 2018–2021). Notably, we focus on advances in biosensors for phosphatidylinositol, phosphatidic acid, and PI 3-kinase lipid products.     

Evaluation of permselective membranes for optimization of intracerebral amperometric glutamate biosensors

Monitoring of extracellular brain glutamate concentrations by intracerebral biosensors is a promising approach to further investigate the role of this important neurotransmitter. However, amperometric biosensors are typically hampered by Faradaic interference caused by the presence of other electroactive species in the brain, such as ascorbic acid, dopamine, and uric acid. Various permselective membranes are often used on biosensors to prevent this. In this study we evaluated the most commonly used membranes, i.e. nafion, polyphenylenediamine, polypyrrole, polyaniline, and polynaphthol using a novel silica-based platinum electrode. First we selected the membranes with the highest sensitivity for hydrogen peroxide in vitro and an optimal selectivity against electrochemical interferents. Then we evaluated the performances of these membranes in a short lasting (3-4h) in vivo experiment. We found that best in vitro performance was accomplished with biosensors that were protected by a poly(m-phenylenediamine) membrane deposited onto the platinum electrode by cyclic voltammetry. However, post-implantation evaluation of these membranes showed poor selectivity against dopamine. Combination with a previously applied nafion layer did not protect the sensors against acute biofouling; indeed it was even counter effective. Finally, we investigated the ability of our biosensors to monitor the effect of glutamate transport blocker DL-TBOA on modulating glutamate concentrations in the prefrontal cortex of anaesthetized rats. The optimized biosensors recorded a rapid 35-fold increase in extracellular glutamate, and are considered suitable for further exploration in vivo.     

Dipstick-type biosensor for visual detection of DNA with oligonucleotide-decorated colored polystyrene microspheres as reporters

In recent years, there is a continuously growing interest in the development of biosensors for rapid, simple and inexpensive DNA tests suitable for the small laboratory or for on-site testing. Detection is accomplished through electrochemical, optical or gravimetric transduction. We report on the development of disposable dipstick-type DNA biosensors that employ oligonucleotide-decorated colored polystyrene microspheres as reporters and enable visual detection of DNA sequences without the use of instrumentation. The biosensors have been designed to detect DNA molecules that contain both, a biotin moiety and a segment that is complementary to the oligonucleotide attached on the surface of blue or red microspheres. Capture of the hybrids by immobilized streptavidin at the test zone results in the formation of a colored line. The biosensors were applied to: (a) detection of single-stranded DNA, (b) detection of PCR-amplified double-stranded DNA and (c) genotyping of single nucleotide polymorphisms (SNP). The results were compared with sensors based on gold nanoparticle reporters. It is also demonstrated that the microspheres offer the potential for multicolor detection of specific DNA sequences.     

Thermostable Proteins as Probe for the Design of Advanced Fluorescence Biosensors

In this review we explore the advantages deriving from the use of either enzymes or sugar binding proteins isolated from thermophilic organisms to develop stable fluorescence biosensors. We report on a novel approach to address the consumption of the analyte by enzyme-based biosensors, namely the utilization of apo-enzymes as non-active forms of proteins which are still able to bind the ligand but cannot transform it into product. We also report recent studies in which the fluorescence labeling of a naturally thermostable binding protein allows a quantitative determination of glucose.This work is dedicated to Prof. Koki Horikoshi for his outstanding contribution to the knowledge of the world of extremophiles.     

Development of an optimized backbone of FRET biosensors for kinases and GTPases

Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have shed new light on the spatiotemporal dynamics of signaling molecules. Among them, intramolecular FRET biosensors have been increasingly used due to their high sensitivity and user-friendliness. Time-consuming optimizations by trial and error, however, obstructed the development of intramolecular FRET biosensors. Here we report an optimized backbone for rapid development of highly sensitive intramolecular FRET biosensors. The key concept is to exclude the “orientation-dependent” FRET and to render the biosensors completely “distance-dependent” with a long, flexible linker. We optimized a pair of fluorescent proteins for distance-dependent biosensors, and then developed a long, flexible linker ranging from 116 to 244 amino acids in length, which reduced the basal FRET signal and thereby increased the gain of the FRET biosensors. Computational simulations provided insight into the mechanisms by which this optimized system was the rational strategy for intramolecular FRET biosensors. With this backbone system, we improved previously reported FRET biosensors of PKA, ERK, JNK, EGFR/Abl, Ras, and Rac1. Furthermore, this backbone enabled us to develop novel FRET biosensors for several kinases of RSK, S6K, Akt, and PKC and to perform quantitative evaluation of kinase inhibitors in living cells.     

The present and the future of protein biosensor engineering

Protein biosensors play increasingly important roles in cell and neurobiology and have the potential to revolutionise the way clinical and industrial analytics are performed. The gradual transition from multicomponent biosensors to fully integrated single chain allosteric biosensors has brought the field closer to commercial applications. We evaluate various approaches for converting constitutively active protein reporter domains into analyte operated switches. We discuss the paucity of the natural receptors that undergo conformational changes sufficiently large to control the activity of allosteric reporter domains. This problem can be overcome by constructing artificial versions of such receptors. The design path to such receptors involves the construction of Chemically Induced Dimerisation systems (CIDs) that can be configured to operate single and two-component biosensors.     

压电生物传感器研究进展

作为一项新兴综合型科学技术,生物传感器是近年来生物化学、分子生物学、传感器技术等领域的研究热点之一。本介绍了压电生物传感器的基本原理、组成、分类,并对近年来国内外的研究进展、生物识别元件的固定化技术以及压电生物传感器的发展趋势作综合评述。