Diagnostic Biosensors Research Articles

Progress and bioapplication of CRISPR-based one-step, quantitative and multiplexed infectious disease diagnostics.

In Vitro Diagnosis (IVD) technology is able to accurately detect pathogens or biomarkers at an initial stage of disease, which works as an important toolbox for disease diagnosis. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system, as an emerging IVD method, plays a crucial role in the field of infectious disease detection due to its superior sensitivity and specificity. Recently, an increasing number of scientists have been devoted to improving the performance of CRISPR-based detection and on-site point-of-care testing (POCT) from extraction-free detection, amplification-free, modified Cas/crRNA complexes, quantitative assays, one-pot detection, and multiplexed platform. In this review, we describe the potential roles of these novel approaches and platforms in one-pot methods, quantitative molecular diagnostics as well as multiplexed detection. This review will not only help guide the full use of the CRISPR-Cas tools for quantification, multiplexed detection, POCT and as next-generation diagnostic biosensing platforms but also inspire new ideas, technological advances, and engineering strategies to address real-world challenges like the ongoing COVID-19 pandemic.

Magnetite-Based Biosensors and Molecular Logic Gates: From Magnetite Synthesis to Application

In the last few decades, point-of-care (POC) sensors have become increasingly important in the detection of various targets for the early diagnostics and treatment of diseases. Diverse nanomaterials are used as building blocks for the development of smart biosensors and magnetite nanoparticles (MNPs) are among them. The intrinsic properties of MNPs, such as their large surface area, chemical stability, ease of functionalization, high saturation magnetization, and more, mean they have great potential for use in biosensors. Moreover, the unique characteristics of MNPs, such as their response to external magnetic fields, allow them to be easily manipulated (concentrated and redispersed) in fluidic media. As they are functionalized with biomolecules, MNPs bear high sensitivity and selectivity towards the detection of target biomolecules, which means they are advantageous in biosensor development and lead to a more sensitive, rapid, and accurate identification and quantification of target analytes. Due to the abovementioned properties of functionalized MNPs and their unique magnetic characteristics, they could be employed in the creation of new POC devices, molecular logic gates, and new biomolecular-based biocomputing interfaces, which would build on new ideas and principles. The current review outlines the synthesis, surface coverage, and functionalization of MNPs, as well as recent advancements in magnetite-based biosensors for POC diagnostics and some perspectives in molecular logic, and it also contains some of our own results regarding the topic, which include synthetic MNPs, their application for sample preparation, and the design of fluorescent-based molecular logic gates.

Silver-coated ZnO disordered nanostructures as low-cost and label-free Raman biosensing platform for fast detection of complex organic profiles

The need of cheap and easy to use biosensors for rapid diagnostics is a crucial challenge in medicine: indeed, devices must offer multiple properties such as fast response, high sensitivity, reduced need of sample processing, etc. In this respect, optical biosensors remain a promising class of biodevices. Here we present an extensive investigation of the surface properties of silver-coated zinc oxide disordered nanostructures as efficient biosensing platform to detect complex organic profiles via Raman spectroscopy. In particular, we combine low temperature and large area manufacturing techniques to offer a low-cost tool for rapid detection of untreated organic samples like human blood avoiding the usage of expensive manufacturing methods to grow ordered nanostructures and providing safer materials respect the implementation of metal nanoparticles thus opening the possibility to adopt these devices also in vivo. After an accurate study using rhodamine, comparing Raman response for as deposited and lasered surfaces, different metal layers (gold vs silver) and different metal thicknesses (from 25 to 150 nm), we successfully validate the performance of the silver-coated lasered platform on untreated human blood obtaining high resolved response and identifying several organic signatures attributable to hypoxanthine, glucose and lipoprotein.

Point-of-Care Diagnostic Biosensors to Monitor Anti-SARS-CoV-2 Neutralizing IgG/sIgA Antibodies and Antioxidant Activity in Saliva.

Monitoring biomarkers is a great way to assess daily physical condition, and using saliva instead of blood samples is more advantageous as the process is simple and allows individuals to test themselves. In the present study, we analyzed the titers of neutralizing antibodies, IgG and secretory IgA (sIgA), in response to the SARS-CoV-2 vaccine, in saliva. A total of 19 saliva and serum samples were collected over a 10-month period 3 weeks after the first vaccine, 8 months after the second vaccine, and 1 month after the third vaccine. The ranges of antibody concentrations post-vaccination were: serum IgG: 81-15,000 U/mL, salivary IgG: 3.4-330 U/mL, and salivary IgA: 58-870 ng/mL. A sharp increase in salivary IgG levels was observed after the second vaccination. sIgA levels also showed an increasing trend. A correlation with trends in serum IgG levels was observed, indicating the possibility of using saliva to routinely assess vaccine efficacy. The electrochemical immunosensor assay developed in this study based on the gold-linked electrochemical immunoassay, and the antioxidant activity measurement based on luminol electrochemiluminescence (ECL), can be performed using portable devices, which would prove useful for individual-based diagnosis using saliva samples.

The successes and challenges in the development of sensors for medical diagnostics

Co-Editors-in-Chief Sabine Szunerits and Xueji Zhang introduce the first anniversary issue of Sensors & Diagnostics by reflecting on state-of-the-art developments in biosensors for medical diagnostics.

Design of an optical nanobiosensor for detection of Legionella pneumophila in water samples.

Legionella spp. is a causative agent of Legionnaires' disease that creates public health problems. Isolation of these bacteria from water sources is essential to identify outbreak origins and prevent disease. Diagnostic biosensors for water quality control to protect consumers from water-borne infections can predict many outbreaks. Gold nanoparticles conjugated probes are a new generation of diagnostic tools. In this study, an optical nano biosensor was designed and characterized to detect Legionella pneumophila in water samples rapidly. Thiolated probes designed for the mip gene were attached to gold nanoparticles and then water samples containing Legionella pneumophila were examined. The limit of detection for PCR and biosensor was 104 and 103 copy numbers/μl, respectively. Biosensor sensitivity and PCR were reported to be 90% (18 out of 20) and 85% (17 out of 20), respectively. Specificity 100% has been reported for both methods. According to the obtained results, this method has the potential to diagnose L. pneumophila with high sensitivity and specificity. This system can be employed as a practical tool for rapid, accurate, high-sensitivity, and acceptable detection of Legionella pneumophila in contaminated water, which is cost-effective in terms of cost and time.

Inflammatory Stimuli Responsive Non-Faradaic, Ultrasensitive Combinatorial Electrochemical Urine Biosensor.

In this work, we propose a novel diagnostic biosensor that can enable stratification of disease states based on severity and hence allow for clear and actionable diagnoses. The scheme can potentially boost current Point-Of-Care (POC) biosensors for diseases that require time-critical stratification. Here, two key inflammatory biomarkers—Interleukin-8 and Interleukin-6—have been explored as proof of concept, and a four-class stratification of inflammatory disease severity is discussed. Our method is superior to traditional lab techniques as it is faster (<4 minutes turn-around time) and can work with any combination of disease biomarkers to categorize diseases by subtypes and severity. At its core, the biosensor relies on electrochemical impedance spectroscopy to transduce subtle inflammatory stimuli at the input for IL-8 and IL-6 for a limit of detection (LOD) of 1 pg/mL each. The biosensing scheme utilizes a two-stage random forest machine learning model for 4-state output disease classification with a 98.437% accuracy. This scheme can potentially boost the diagnostic power of current electrochemical biosensors for better precision therapy and improved patient outcomes.

Recent Advances of Representative Optical Biosensors for Rapid and Sensitive Diagnostics of SARS-CoV-2.

The outbreak of Corona Virus Disease 2019 (COVID-19) has again emphasized the significance of developing rapid and highly sensitive testing tools for quickly identifying infected patients. Although the current reverse transcription polymerase chain reaction (RT-PCR) diagnostic techniques can satisfy the required sensitivity and specificity, the inherent disadvantages with time-consuming, sophisticated equipment and professional operators limit its application scopes. Compared with traditional detection techniques, optical biosensors based on nanomaterials/nanostructures have received much interest in the detection of SARS-CoV-2 due to the high sensitivity, high accuracy, and fast response. In this review, the research progress on optical biosensors in SARS-CoV-2 diagnosis, including fluorescence biosensors, colorimetric biosensors, Surface Enhancement Raman Scattering (SERS) biosensors, and Surface Plasmon Resonance (SPR) biosensors, was comprehensively summarized. Further, promising strategies to improve optical biosensors are also explained. Optical biosensors can not only realize the rapid detection of SARS-CoV-2 but also be applied to judge the infectiousness of the virus and guide the choice of SARS-CoV-2 vaccines, showing enormous potential to become point-of-care detection tools for the timely control of the pandemic.

A Polyclonal SELEX Aptamer Library Allows Differentiation of Candida albicans, C. auris and C. parapsilosis Cells from Human Dermal Fibroblasts.

Easy and reliable identification of pathogenic species such as yeasts, emerging as problematic microbes originating from the genus Candida, is a task in the management and treatment of infections, especially in hospitals and other healthcare environments. Aptamers are seizing an already indispensable role in different sensing applications as binding entities with almost arbitrarily tunable specificities and optimizable affinities. Here, we describe a polyclonal SELEX library that not only can specifically recognize and fluorescently label Candida cells, but is also capable to differentiate C. albicans, C. auris and C. parapsilosis cells in flow-cytometry, fluorometric microtiter plate assays and fluorescence microscopy from human cells, exemplified here by human dermal fibroblasts. This offers the opportunity to develop diagnostic tools based on this library. Moreover, these specific and robust affinity molecules could also serve in the future as potent binding entities on biomaterials and as constituents of technical devices and will thus open avenues for the development of cost-effective and easily accessible next generations of electronic biosensors in clinical diagnostics and novel materials for the specific removal of pathogenic cells from human bio-samples.

Current Perspectives in Graphene Oxide-Based Electrochemical Biosensors for Cancer Diagnostics.

Since the first commercial biosensor device for blood glucose measurement was introduced in the 1970s, many “biosensor types” have been developed, and this research area remains popular worldwide. In parallel with some global biosensor research reports published in the last decade, including a great deal of literature and industry statistics, it is predicted that biosensor design technologies, including handheld or wearable devices, will be preferred and highly valuable in many areas in the near future. Biosensors using nanoparticles still maintain their very important place in science and technology and are the subject of innovative research projects. Among the nanomaterials, carbon-based ones are considered to be one of the most valuable nanoparticles, especially in the field of electrochemical biosensors. In this context, graphene oxide, which has been used in recent years to increase the electrochemical analysis performance in biosensor designs, has been the subject of this review. In fact, graphene is already foreseen not only for biosensors but also as the nanomaterial of the future in many fields and is therefore drawing research attention. In this review, recent and prominent developments in biosensor technologies using graphene oxide (GO)-based nanomaterials in the field of cancer diagnosis are briefly summarized.

Y-shaped DNA nanostructures assembled-spherical nucleic acids as target converters to activate CRISPR-Cas12a enabling sensitive ECL biosensing

Considering the trans-cleavage capabilities, high-specificity and programmability, the CRISPR-Cas system has been recognized as a valuable platform to develop the next-generation diagnostic biosensors. However, due to the natural interaction with nucleic acids, current CRISPR-Cas-based detection mostly applies in nucleic acid analysis rather than non-nucleic acid analysis. By virtue of spherical nucleic acids (SNAs) with programmability and specificity, the Y-shaped DNA nanostructures assembled-SNAs (Y–SNAs) were rationally designed as target converters to achieve the quantitative activation of CRISPR-Cas12a, enabling a highly specific and sensitive electrochemiluminescence (ECL) determination of alpha-methylacyl-CoA racemase (AMACR), a high specific protein biomarker of prostate cancer. Significantly, the Y-shaped DNA nanostructures comprised of assisted DNA (A1), AMACR aptamer and DNA activator of CRISPR-Cas12a were loaded on Au nanoparticles modified Fe3O4 magnetic beads (Au@Fe3O4 MBs) to construct the robust Y–SNAs. In the presence of the target AMACR, the Y–SNAs as target converters could achieve quantitative activation of CRISPR-Cas12a by outputting the DNA activators with a linear relationship to the target. The amplified ECL signals were triggered by the release of the ferrocene-labeled quenching probes (QPs) on the electrode surface due to the trans-cleavage activity of CRISPR-Cas12a, thereby realizing the sensitive ECL determination of AMACR from 10 ng/mL to 100 μg/mL with the detection limit of 1.25 ng/mL. In general, this approach provides novel perspectives on how to design a universal ECL platform of the CRISPR-Cas system to detect the non-nucleic acid targets beyond the traditional methods.

A concise review on potential cancer biomarkers and advanced manufacturing of smart platform-based biosensors for early-stage cancer diagnostics

Advanced diagnostic technologies for early-stage detection of cancer are essential for proper management and guided therapeutics to increase chances of survival for patients. Presently, most forms of cancer get diagnosed post metastasis throughout the body resulting in late-stage treatment leading to fatality which may have otherwise been avoided. In order to overcome this limitation in cancer management, accurate, rapid and effective clinical screening diagnostic methods for cancer detection need to be urgently developed. In this review, we have compiled the extensive research being carried out in recent times on fabrication of biosensors for cancer diagnostics, as these devices may be customised to detect a particular target bio analyte (disease biomarker) of a specific cancer type, with the help of a corresponding bioreceptor (i.e., nucleic acid, protein, enzyme) using electrochemical/optical/piezoelectric signals that can be analysed with a special emphasis on the latest advancement in Internet of Things (IoT) based sensing technologies. We have correspondingly enumerated the latest cancer biomarkers that have been identified and may be used for potential diagnosis as well as to determine the effectiveness of drugs at different target sites and anticancer chemotherapeutic medication using sensor-based technology. This review provides a restructured base for ongoing research on cancer diagnostics, which may potentially help develop accurate point-of-care rapid diagnostic screening methods, for effective monitoring, management and treatment of angiogenesis and cancer metastasis at an early stage and reduce fatalities.

An Electrochemical Biosensor for Detection of P.69 Pertactin Associated with B. pertussis

Whooping cough caused by Bordetella pertussis can cause serious and prolonged health effects and is especially deadly in infants. Early treatment can significantly reduce the duration of illness and lead to better outcomes, but current diagnostic procedures need a relatively large patient sample and have an extended wait period for testing results. There is thus a pressing need for quick and accurate diagnosis. In response, we are developing an electrochemical DNA‐based (E‐DNA) biosensor for the rapid detection of B. pertussis. Our biosensor utilizes a DNA aptamer that targets P.69 pertactin, a well‐known adhesion factor in the outer membrane of B. pertussis. To generate this aptamer, we first obtained purified P.69 pertactin and biotinylated it. Biotinylation extent was measured via HABA assay and showed ~ 42 biotin molecules per P.69 pertactin molecule. This biotinylated protein was used for selection in a modified SELEX (systematic evolution of ligands by exponential enrichment) process, followed by high‐throughput sequencing. That analysis discovered six aptamer sequences with potential strong binding for P.69 pertactin. Potential aptamers were synthesized, and binding was confirmed via fluorescence anisotropy and gel shift assays. These techniques identified one aptamer sequence as having strong, low‐nanomolar binding affinity for P.69 pertactin. This aptamer was then computationally analyzed for likely secondary and tertiary structures, allowing identification of the minimum binding unit, and then was modified to generate a two‐state binding system. This modified aptamer sequence serves as the basis for an electrode‐tethered E‐DNA biosensor. This design will be verified against purified P.69 pertactin standards as well as B. pertussis isolates to demonstrate its potential as a rapid and accurate diagnostic biosensor for whooping cough infections.

Designing a CRISPR/Cas12a- and Au-Nanobeacon-Based Diagnostic Biosensor Enabling Direct, Rapid, and Sensitive miRNA Detection

Direct, rapid, sensitive, and selective detection of nucleic acids in complex biological fluids is crucial for medical early diagnosis. We herein combine the trans-cleavage ability of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a with Au-nanobeacon to establish a CRISPR-based biosensor, providing rapid miRNA detection with high speed and attomolar sensitivity. In this strategy, we first report that the trans-cleavage activity of CRISPR/cas12a, which was previously reported to be triggered only by target ssDNA or dsDNA, can be activated by the target miRNA directly. Therefore, this method is direct, i.e., does not need the conversion of miRNA into its complementary DNA (cDNA). Meanwhile, as compared to the traditional ssDNA reporters and molecular beacon (MB) reporters, the Au-nanobeacon reporters exhibit improved reaction kinetics and sensitivity. In this assay, the miRNA-21 could be detected with very high sensitivity in only 5 min. Finally, the proposed strategy enables rapid, sensitive, and selective miRNA determination in complex biological samples, providing a potential tool for medical early diagnosis.

ISOLATION, SCREENING, AND PARTIAL CHARACTERIZATION OF OXALATE OXIDASE ENZYME FROM PSEUDOMONAS STRAIN UNDER INDUCED OXALATE STRESS CONDITION

Oxalate oxidase (OxO), a manganese dependent enzyme, is involved in the catalysis of oxalate oxidation to carbon dioxide with the formation of hydrogen peroxide. OxO is present in the cell wall of plants. It increases the resistivity against diseases and external stress. Oxalate is found as waste metabolites in mammals, excess accumulation of oxalate results in hyperoxaluria and urolithiasis in humans. These disorders can be diagnosed by the help of OxO in many analytical methods. The present study is aimed on isolation of OxO enzyme from Pseudomonas strain under high salt stress. The OxO enzyme was produced in bulk under selected salt stress. It was partially purified by ammonium sulphate precipitation method, dialysis and ion-exchange chromatography. The OxO enzyme on enzymatic analysis showed potent activity concerning oxalic acid substrate. The microtiter plate analysis confirmed the ability of OxO enzyme purified from Pseudomonas strain in the diagnosis of oxalate-related disorders and in the future could be used to prepare the diagnostic biosensors.

Thin flexible lab-on-a-film for impedimetric sensing in biomedical applications

Microfluidic cytometers based on coulter principle have recently shown a great potential for point of care biosensors for medical diagnostics. Here, we explore the design of an impedimetric microfluidic cytometer on flexible substrate. Two coplanar microfluidic geometries are compared to highlight the sensitivity of the device to the microelectrode positions relative to the detection volume. We show that the microelectrodes surface area and the geometry of the sensing volume for the cells strongly influence the output response of the sensor. Reducing the sensing volume decreases the pulse width but increases the overall pulse amplitude with an enhanced signal-to-noise ratio (~ max. SNR = 38.78 dB). For the proposed design, the SNR was adequate to enable good detection and differentiation of 10 µm diameter polystyrene beads and leukemia cells (~ 6–21 µm). Also, a systematic approach for irreversible & strong bond strength between the thin flexible surfaces that make up the biochip is explored in this work. We observed the changes in surface wettability due to various methods of surface treatment can be a valuable metric for determining bond strength. We observed permanent bonding between microelectrode defined polypropylene surface and microchannel carved PDMS due to polar/silanol groups formed by plasma treatment and consequent covalent crosslinking by amine groups. These experimental insights provide valuable design guidelines for enhancing the sensitivity of coulter based flexible lab-on-a-chip devices which have a wide range of applications in point of care diagnostics.

Label-Free Physical Techniques and Methodologies for Proteins Detection in Microfluidic Biosensor Structures.

Proteins in biological fluids (blood, urine, cerebrospinal fluid) are important biomarkers of various pathological conditions. Protein biomarkers detection and quantification have been proven to be an indispensable diagnostic tool in clinical practice. There is a growing tendency towards using portable diagnostic biosensor devices for point-of-care (POC) analysis based on microfluidic technology as an alternative to conventional laboratory protein assays. In contrast to universally accepted analytical methods involving protein labeling, label-free approaches often allow the development of biosensors with minimal requirements for sample preparation by omitting expensive labelling reagents. The aim of the present work is to review the variety of physical label-free techniques of protein detection and characterization which are suitable for application in micro-fluidic structures and analyze the technological and material aspects of label-free biosensors that implement these methods. The most widely used optical and impedance spectroscopy techniques: absorption, fluorescence, surface plasmon resonance, Raman scattering, and interferometry, as well as new trends in photonics are reviewed. The challenges of materials selection, surfaces tailoring in microfluidic structures, and enhancement of the sensitivity and miniaturization of biosensor systems are discussed. The review provides an overview for current advances and future trends in microfluidics integrated technologies for label-free protein biomarkers detection and discusses existing challenges and a way towards novel solutions.

Hybrid-Integrated Biosensor for Express Determination of Protein Markers of Diseases based on Molecular Recognition and Direct Fluorimetric Detection

Ways of creating new generation biosensors for multiparametric express diagnostics based on molecular recognition and direct fluorimetric registration of a peptide aptamer — protein marker complex were considered. The biosensor platform comprises a microfluidic channel for delivery sample solutions, coupled with flow-through zones containing covalently attached arrays of peptide probes — aptamers. An outer glass window of the biochip assembly contains a layer of luminophore ZnS:Cu, bound on it via an acrylic lacquer and intended for the re-emitting native fluorescence of bound proteins into the longer wavelength range, more efficient in registering signals with CMOS sensors. The aptamers were designed using "Protein 3D" program for analysis of spatial complementarity of protein structures. The peptide, complementary to Troponin T, was modified by replacement of aromatic amino acid residue while maintaining the spatial configuration. The complementarity of peptide and Troponin T was confirmed using a capillary electrophoresis-on-a-chip. Biosensors are manufactured using thick-film technology and photolithography. The fluorescence of marker proteins was excited using UV-LED with a radiation wavelength of 275 nm. The limit of detection achieved for Troponin T was 6 ng/ml.

ISOLATION, SCREENING, AND PARTIAL CHARACTERIZATION OF OXALATE OXIDASE ENZYME FROM PSEUDOMONAS STRAIN UNDER INDUCED OXALATE STRESS CONDITION

Oxalate oxidase (OxO), a manganese dependent enzyme, is involved in the catalysis of oxalate oxidation to carbon dioxide with the formation of hydrogen peroxide. OxO is present in the cell wall of plants. It increases the resistivity against diseases and external stress. Oxalate is found as waste metabolites in mammals, excess accumulation of oxalate results in hyperoxaluria and urolithiasis in humans. These disorders can be diagnosed by the help of OxO in many analytical methods. The present study is aimed on isolation of OxO enzyme from Pseudomonas strain under high salt stress. The OxO enzyme was produced in bulk under selected salt stress. It was partially purified by ammonium sulphate precipitation method, dialysis and ion-exchange chromatography. The OxO enzyme on enzymatic analysis showed potent activity concerning oxalic acid substrate. The microtiter plate analysis confirmed the ability of OxO enzyme purified from Pseudomonas strain in the diagnosis of oxalate-related disorders and in the future could be used to prepare the diagnostic biosensors.

Optical Biosensors for Diagnostics of Infectious Viral Disease: A Recent Update.

The design and development of biosensors, analytical devices used to detect various analytes in different matrices, has emerged. Biosensors indicate a biorecognition element with a physicochemical analyzer or detector, i.e., a transducer. In the present scenario, various types of biosensors have been deployed in healthcare and clinical research, for instance, biosensors for blood glucose monitoring. Pathogenic microbes are contributing mediators of numerous infectious diseases that are becoming extremely serious worldwide. The recent outbreak of COVID-19 is one of the most recent examples of such communal and deadly diseases. In efforts to work towards the efficacious treatment of pathogenic viral contagions, a fast and precise detection method is of the utmost importance in biomedical and healthcare sectors for early diagnostics and timely countermeasures. Among various available sensor systems, optical biosensors offer easy-to-use, fast, portable, handy, multiplexed, direct, real-time, and inexpensive diagnosis with the added advantages of specificity and sensitivity. Many progressive concepts and extremely multidisciplinary approaches, including microelectronics, microelectromechanical systems (MEMSs), nanotechnologies, molecular biology, and biotechnology with chemistry, are used to operate optical biosensors. A portable and handheld optical biosensing device would provide fast and reliable results for the identification and quantitation of pathogenic virus particles in each sample. In the modern day, the integration of intelligent nanomaterials in the developed devices provides much more sensitive and highly advanced sensors that may produce the results in no time and eventually help clinicians and doctors enormously. This review accentuates the existing challenges engaged in converting laboratory research to real-world device applications and optical diagnostics methods for virus infections. The review’s background and progress are expected to be insightful to the researchers in the sensor field and facilitate the design and fabrication of optical sensors for life-threatening viruses with broader applicability to any desired pathogens.