Development of a fluorescent scaffold by utilizing quercetin template for selective detection of Hg2+: Experimental and theoretical studies along with live cell imaging.

Quercetin is an important antioxidant with high bioactivity and it has been used as SARS-CoV-2 inhibitor significantly. Quercetin, one of the most abundant flavonoids in nature, has been in the spot of numerous experimental and theoretical studies in the past decade due to its great biological and medicinal importance. But there have been limited instances of employing quercetin and its derivatives as a fluorescent framework for specific detection of various cations and anions in the chemosensing field. Therefore, we have developed a novel chemosensor based on quercetin coupled benzyl ethers (QBE) for selective detection of Hg2+ with "naked-eye" colorimetric and "turn-on" fluorometric response. Initially QBE itself exhibited very weak fluorescence with low quantum yield (Φ = 0.009) due to operating photoinduced electron transfer (PET) and inhibition of excited state intramolecular proton transfer (ESIPT) as well as intramolecular charge transfer (ICT) within the molecule. But in presence of Hg2+, QBE showed a sharp increase in fluorescence intensity by 18-fold at wavelength 444 nm with high quantum yield (Φ = 0.159) for the chelation-enhanced fluorescence (CHEF) with coordination of Hg2+, which hampers PET within the molecule. The strong binding affinity of QBE towards Hg2+ has been proved by lower detection limit at 8.47 µM and high binding constant value as 2 × 104 M-1. The binding mechanism has been verified by DFT study, Cyclic voltammograms and Jobs plot analysis. For the practical application, the binding selectivity of QBE with Hg2+ has been capitalized in physiological medium to detect intracellular Hg2+ levels in living plant tissue by using green gram seeds. Thus, employing QBE as a fluorescent chemosensor for the specific identification of Hg2+ will pave the way for a novel approach to simplifying the creation of various chemosensors based on quercetin backbone for the precise detection of various biologically significant analytes.

A Redox Mediator-Free Highly Selective and Sensitive Electrochemical Aptasensor for Patulin Mycotoxin Detection in Apple Juice Using Ni-NiO Pseudocapacitive Nanomaterials.

Pseudocapacitive nanomaterials have recently gained significant attention in electrochemical biosensors due to their rapid response, long cycle life, high surface area, biomolecule compatibility, and superior energy storage capabilities. In our study, we introduce the potential of using Ni-NiO nanofilm's pseudocapacitive traits as transducer signals in electrochemical aptasensors. Capitalizing on the innate affinity between histidine and nickel, we immobilized histidine-tagged streptavidin (HTS) onto Ni-NiO-modified electrodes. Additionally, we employed a biolayer interferometry-based SELEX to generate biotinylated patulin aptamers. These aptamers, when placed on Ni-NiO-HTS surfaces, make a suitable biosensing platform for rapid patulin mycotoxin detection in apple juice using electrochemical amperometry in microseconds. The novelty lies in optimizing pseudocapacitive nanomaterials structurally and electrochemically, offering the potential for redox mediator-free electrochemical aptasensors. Proof-of-concept is conducted by applying this surface for the ultrasensitive detection of a model analyte, patulin mycotoxin. The aptamer-functionalized bioelectrode showed an excellent linear response (10-106 fg/mL) and an impressive detection limit (1.65 fg/mL, +3σ of blank signal). Furthermore, reproducibility tests yielded a low relative standard deviation of 0.51%, indicating the good performance of the developed biosensor. Real sample analysis in freshly prepared apple juice revealed no significant difference (P < 0.05) in current intensity between spiked and real samples. The sensor interface maintained excellent stability for up to 2 weeks (signal retention 96.45%). The excellent selectivity, stability, and sensitivity of the electrochemical aptasensor exemplify the potential for using nickel-based pseudocapacitive nanomaterials for a wide variety of electrochemical sensing applications.

A nanobody based ultrasensitive electrochemical biosensor for the detection of soluble CTLA-4 -A candidate biomarker for cancer development and progression.

A soluble isoform of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) has been found in the serum of healthy individuals and alterations in its expression level have been linked with the development and progression of various cancers. Conventionally, soluble CTLA-4 (sCTLA-4) has been quantified by techniques such as ELISA, western blot, and flow cytometry, which however are time-consuming, highly expensive and require large sample volumes. Therefore, rapid, cost-effective and real-time monitoring of soluble CTLA-4 levels is much needed to facilitate timely diagnosis of a worsening disease and help patient selection for immunotherapeutic interventions in cancer. Here, for the first time, we report an ultrasensitive, highly selective electrochemical nanobody (NAb) based biosensor for the quantitative detection of soluble CTLA-4 employing poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) and gold nanoparticles modified electrode with attomole sensitivity. Incorporating nanomaterials with conductive polymers enhances the sensitivity of the electrochemical biosensor, while the nanobody's stability, specificity and ease of production make it a suitable choice as a bioreceptor. The proposed NAb-based sensor can detect sCTLA-4 from pure recombinant protein in a wide concentration range of 100 ag mL-1- 500 µg mL-1, with a limit of detection of 1.19 ag mL-1 (+3σ of the blank signal). The sensor's relative standard deviation for reproducibility is less than 0.4% and has effective real sample analytics for cell culture supernatant with no significant difference with pure recombinant protein (p < 0.05). Our proposed nanobody based sensor exhibits stability for up to 2 weeks (<3% variation). Moreover, this nanobody-based sensor presents a future opportunity for quantitative, ultrasensitive, and economical biosensor development that can be adapted to monitor the immune landscape of cancer patients to provide a larger therapeutic window.

A smartphone-integrated low-cost, reagent-free, non-destructive dried blood spot-based paper sensor for hematocrit measurement.

The blood hematocrit (Hct) level provides vital information about a person's health. Traditional Hct measurement equipment relies heavily on infrastructure and skilled manpower, limiting its broad implementation in resource-limited contexts. Therefore, we developed a simple, reagent-free, non-destructive, smartphone-integrated paper-based device for Hct measurement by analyzing blood-spreading area on a paper substrate. Blood spreading area was found to be dependent on the Hct value, paper properties, and assay duration. This device was calibrated using a custom-made Python algorithm with 10 µl of blood, which produced a sensitivity of -1.90 ± 0.03 mm2/Hct (%) with a LOD as low as 2.17% Hct. The device linear range (8.8 to 58% Hct) is wide enough to cover the clinically relevant range of blood Hct (%). Furthermore, this Python algorithm was coupled with a user-friendly and clinically beneficial Android application (app) to establish an automated tool for quantitative estimation. Comparing the app performance with the result obtained from the gold standard hematology analyzer using blood from 87 subjects reveals a strong correlation (r = 0.99), an average bias of 0.15 with limits of agreement of -2.5 to 2.79 at 95% CI. The device exhibits an accuracy of 96.85% and acceptable reproducibility, with CV ranging from 0.8 to 7.5%. An integrated detection cum readout guiding pattern may allow this device to be suitable for simultaneous quantitative and qualitative estimation and to be employed in both developed and resource-limited clinical settings for Hct measurement in routine checkups and regular monitoring during critical care, as well as in the initial screening of large anemic populations.

A fast survey on recent developments in designing colorimetric and fluorescent sensors for the selective detection of essential amino acids.

Owing to the biological significance of various amino acids, developing accurate and cost-effective sensing techniques for the selective detection of amino acids has recently attracted growing interest. This review discusses the recent advancements of chemosensors in the selective detection of only essential amino acids out of a total of twenty amino acids, which have been applied in chemosensing research, and the mechanism of their action. The focus is directed towards the detection of the most important essential amino acids, like leucine, threonine, lysine, histidine, tryptophan and methionine, since isoleucine and valine are yet to be explored in regard to chemosensing. According to their chemical and fluorescence properties, different sensing techniques, such as the reaction-based approach, DNA-based sensors, nanoparticle formation, coordination ligand binding, host-guest chemistry, the fluorescence indicator displacement (FID) approach, electrochemical sensors, carbon dot-based sensors, MOF-based sensors and metal-based techniques, have been described.

Immunological profiling and development of a sensing device for detection of IL-13 in COPD and asthma.

Chronic obstructive pulmonary disease (COPD) and asthma are the two most common obstructive lung diseases which affects millions worldwide and impose an enormous burden on global healthcare. The overlapping features shared by these two diseases often make differential diagnosis difficult to achieve, leading to misdiagnosis of these patients. Both asthma and COPD are associated with chronic inflammation of the airways which is perpetuated by the interplay between immunological mediators. The crucial role played by these mediators make them attractive targets for disease diagnosis. The present study investigates the immunological mediator profile in these patients as compared with controls. Further, a potential biomarker for the development of a sensing platform is identified. Multiplexed analysis of 8 commonly studied immunological markers (IL-4, IL-5, IL-6, IL-13, TGF-ß, IFN-γ, MCP-1 and NGAL) in serum showed distinct dysregulation pattern, with IL-13 showing the highest potential for differential diagnosis. An impedimetric self-assembled monolayer (SAM) based sensor for detecting IL-13 is developed to distinguish between asthma and COPD. The device shows reliable output with high accuracy and sensitivity towards the detection of IL-13.

Aptamer-based biosensors and their implications in COVID-19 diagnosis.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a novel infectious member of the coronavirus family, has caused millions of cases of infection and deaths all over the world, and been declared a pandemic by the World Health Organization. Conventional laboratory-based diagnostic testing has faced extreme difficulties in meeting the overwhelming demand for testing worldwide, and this has brought about a pressing need for cost-effective rapid diagnosis. There has been a surge in the number of prototypes of diagnostic kits developed, although many of these have been found to be lacking in terms of their accuracy and sensitivity. One type of chip-based diagnostic platform is the aptamer-based biosensor. Aptamers are artificially synthesized oligonucleotides that are capable of specifically binding to a target antigen. As of now, some aptamers have been reported for SARS-CoV-2. Although many ultrasensitive aptasensors have been developed for viruses, few have been successfully adapted for SARS-CoV-2 detection. Our review discusses the recent developments in the domain of SARS-CoV-2 specific aptamer isolation, the design of electrochemical and optical aptasensors, and the implications of aptasensor-based COVID-19 diagnosis.

A comprehensive review on current COVID-19 detection methods: From lab care to point of care diagnosis.

Without a doubt, the current global pandemic affects all walks of our life. It affected almost every age group all over the world with a disease named COVID-19, declared as a global pandemic by WHO in early 2020. Due to the high transmission and moderate mortality rate of this virus, it is also regarded as the panic-zone virus. This potentially deadly virus has pointed up the significance of COVID-19 research. Due to the rapid transmission of COVID-19, early detection is very crucial. Presently, there are different conventional techniques are available for coronavirus detection like CT-scan, PCR, Sequencing, CRISPR, ELISA, LFA, LAMP. The urgent need for rapid, accurate, and cost-effective detection and the requirement to cut off shortcomings of traditional detection methods, make scientists realize to advance new technologies. Biosensors are one of the reliable platforms for accurate, early diagnosis. In this article, we have pointed recent diagnosis approaches for COVID-19. The review includes basic virology of SARS-CoV-2 mainly clinical and pathological features. We have also briefly discussed different types of biosensors, their working principles, and current advancement for COVID-19 detection and prevention.

Electrochemical Biosensors for Cytokine Profiling: Recent Advancements and Possibilities in the Near Future.

Cytokines are soluble proteins secreted by immune cells that act as molecular messengers relaying instructions and mediating various functions performed by the cellular counterparts of the immune system, by means of a synchronized cascade of signaling pathways. Aberrant expression of cytokines can be indicative of anomalous behavior of the immunoregulatory system, as seen in various illnesses and conditions, such as cancer, autoimmunity, neurodegeneration and other physiological disorders. Cancer and autoimmune diseases are particularly adept at developing mechanisms to escape and modulate the immune system checkpoints, reflected by an altered cytokine profile. Cytokine profiling can provide valuable information for diagnosing such diseases and monitoring their progression, as well as assessing the efficacy of immunotherapeutic regiments. Toward this goal, there has been immense interest in the development of ultrasensitive quantitative detection techniques for cytokines, which involves technologies from various scientific disciplines, such as immunology, electrochemistry, photometry, nanotechnology and electronics. This review focusses on one aspect of this collective effort: electrochemical biosensors. Among the various types of biosensors available, electrochemical biosensors are one of the most reliable, user-friendly, easy to manufacture, cost-effective and versatile technologies that can yield results within a short period of time, making it extremely promising for routine clinical testing.

Enzyme-assisted glucose quantification for a painless Lab-on-PCB patch implementation.

In the context of an integrated Lab-on-PCB wearable patch extracting interstitial fluid from the patient via integrated microneedles, the requirements from the integrated biosensing part are quite special compared to static glucose electrochemical biosensors. Hence, in this study, a fully PCB-integrated enzymatic glucose quantification Lab-on-Chip device is presented and evaluated considering these special requirements for such a patch implementation: a) range and limit of detection compatible with interstitial fluid glucose levels of diabetic patients and b) effect of sample flow rate on the biosensing platform performance. This work employs a chronoamperometric approach for glucose detection based on covalently immobilized glucose oxidase on PCB-integrated electrodes. The chronoamperometric measurements show that this platform exhibits µM range sensitivity, high specificity, and good reproducibility, and the assay can detect glucose from 10 µM to 9 mM with a lower limit of detection of 10 µM. The demonstrated detection range under continuous flow proved compatible with interstitial fluid glucose levels of diabetic patients. The sample-to-answer time of our Lab-on-PCB device is less than 1 min (sample delivery of few seconds and 20 s for electrochemical measurement), employing sample volumes of 50 µL in this instance. Increased flow rates substantially improve the platform sensitivity (1.1 µA/mM @0 µL/min to 6.2 µA/mM @10 µL/min), with the measured current increasing exponentially to the flow rate, as opposed to the theoretically expected much lower dependence. This work demonstrates the feasibility of Lab-on-PCB patches in terms of biosensing performance, paving the way for the first cost-effective, painless diabetes management microsystem.

Label-Free Electrochemical Detection of S. mutans Exploiting Commercially Fabricated Printed Circuit Board Sensing Electrodes.

This paper reports for the first time printed-circuit-board (PCB)-based label-free electrochemical detection of bacteria. The demonstrated immunosensor was implemented on a PCB sensing platform which was designed and fabricated in a standard PCB manufacturing facility. Bacteria were directly captured on the PCB sensing surface using a specific, pre-immobilized antibody. Electrochemical impedance spectra (EIS) were recorded and used to extract the charge transfer resistance (Rct) value for the different bacteria concentrations under investigation. As a proof-of-concept, Streptococcus mutans (S. mutans) bacteria were quantified in a phosphate buffered saline (PBS) buffer, achieving a limit of detection of 103 CFU/mL. Therefore, the proposed biosensor is an attractive candidate for the development of a simple and robust point-of-care diagnostic platform for bacteria identification, exhibiting good sensitivity, high selectivity, and excellent reproducibility.

Wash-free, label-free immunoassay for rapid electrochemical detection of PfHRP2 in whole blood samples.

Currently, the diagnosis of many diseases relies on laboratory-based immunoassays (ELISA, Western Blot), which are laborious, time-consuming and expensive. To address these limitations, we report a wash-free and label-free electrochemical immunoassay for rapid measurements of protein biomarkers in blood samples. This immunosensor employs a unique detection scheme based on electrochemical-chemical (EC) redox cycling for signal amplification combined with an affinity-based protein quantification strategy. All of the reagents required for this assay are dried and stored on a stacked membrane assembly, consisting of a Vivid Plasma Separation membrane and two cellulose membranes situated above the sensor, enabling excellent stability at room temperature for up to 2 months. Proof of concept was carried out by performing measurements of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) in whole blood samples, which could be detected from 100 ng/mL to 100 µg/mL with excellent specificity and reproducibility. Each measurement requires only two liquid dispensing steps and can completed in 5 min, making this diagnostic platform promising for point-of-care testing in resource-limited settings.

An ultrasensitive enzyme-free electrochemical immunosensor based on redox cycling amplification using methylene blue.

We report a new enzyme-free electrochemical sensor for ultrasensitive measurements of protein biomarkers in plasma and whole blood samples based on a unique electrochemical-chemical-chemical (ECC) redox cycling signal amplification scheme. This scheme uses methylene blue (MB) as a redox indicator which undergoes an endergonic reaction with Ru(NH3)63+ and a highly exergonic reaction with tris(2-carboxyethyl)phosphine (TCEP). This approach offers improved detection sensitivity and sensor stability compared with enzyme-based ECC redox cycling techniques, while involving a simpler sensor modification process and detection protocol. This redox cycling scheme was combined with a robust immunosandwich assay for quantitative measurements of protein biomarkers. For proof of principle, Plasmodium falciparum histidine-rich protein 2 (PfHRP2) was measured in human plasma and whole blood samples, which could be detected down to 10 fg mL-1 and 18 fg mL-1, respectively. Furthermore, this immunosensor exhibits high selectivity, excellent reproducibility and good stability for up to 2 weeks, making it a promising platform for point-of-care testing, especially for detecting extremely low biomarker concentrations in raw biofluids.

Enzyme-free electrochemical immunosensor based on methylene blue and the electro-oxidation of hydrazine on Pt nanoparticles.

Enzyme-free electrochemical sensors enable rapid, high sensitivity measurements without the limitations associated with enzyme reporters. However, the performance of non-enzymatic electrochemical sensors tends to suffer from slow electrode kinetics and poor signal stability. We report a new enzyme-free electrochemical immunosensor based on a unique competitive detection scheme using methylene blue (MB), hydrazine and platinum nanoparticles (Pt NPs). This scheme is coupled with a robust immunosandwich format employing a MB-labelled detection antibody as a non-enzymatic reporter. In the presence of the target antigen, surface-immobilized MB consumes interfacial hydrazine thereby diminishing the electro-oxidation of hydrazine on Pt NPs. Thus, the concentration of the antigen is directly proportional to the reduction in the electrochemical signal. For proof-of-concept, this sensor was used to detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), an important malaria biomarker, in unadulterated human saliva samples. Chronocoulometric measurements showed that this platform exhibits pM-range sensitivity, high specificity and good reproducibility, making it well suited for many biosensing applications including noninvasive diagnostic testing.

A concentration dependent auto-relay-recognition by the same analyte: a dual fluorescence switch-on by hydrogen sulfide via Michael addition followed by reduction and staining for bio-activity.

H2S is shown, for the first time, to play an extraordinary dual role due to its nucleophilicity and reducing property with our single chemosensor, PND [4-(piperidin-1-yl) naphthalene-1,2-dione]. The initial nucleophilic attack via Michael addition (a lower concentration of H2S, blue fluorescence) is followed by the reduction of the 1,2-diketo functionality (a higher concentration of H2S, green fluorescence). This chemosensor, which also shows biological response, is remarkably effective in sensing the same analyte (H2S) at its different concentrations in a relay pathway via a fluorescence "off-on-on" mechanism, and this is also supported by DFT calculation and Cyclic voltammograms.

Redox cycling-amplified enzymatic Ag deposition and its application in the highly sensitive detection of creatine kinase-MB.

This communication reports a novel enzymatic Ag-deposition scheme combined with chemical-chemical redox cycling by reduced ß-nicotinamide adenine dinucleotide. This novel scheme allows a higher Ag-deposition rate than a scheme using only enzymatic Ag deposition. Therefore, it can be applied for the highly sensitive detection of a cardiac biomarker, creatine kinase-MB.

Sensitive electrochemical detection of vaccinia virus in a solution containing a high concentration of L-ascorbic acid.

Washing processes cannot fully remove interfering species that remain on biosensing surfaces when a sample solution contains a high concentration of interfering species. This study reports an immunosensing scheme employing electroreduction-based electrochemical-chemical (EC) redox cycling that allows sensitive detection of vaccinia virus (VV) in a solution containing a high concentration of L-ascorbic acid (AA). To obtain high signal amplification, an enzymatic reaction by ß-D-galactosidase (Gal) is combined with electroreduction-based EC redox cycling by an oxidant. Among the four possible oxidants (KIO3, NaClO, Ag2O, and H2O2), KIO3 shows the highest signal-to-background ratio and is chosen. During an incubation period of 10 min, Gal converts ß-D-galactopyranoside into p-aminophenol (AP), which is oxidized to p-quinone imine (QI) by KIO3. When -0.05 V vs. Ag/AgCl is applied to an immunosensing electrode, QI is reduced to AP, and the regenerated AP is then reoxidized by KIO3. The electroreduction-based EC redox cycling is induced. An indium-tin oxide electrode modified with reduced graphene oxide and an applied potential of -0.05 V are used to achieve low and reproducible background currents, slow O2 reduction, and fast electroreduction of QI. KIO3 favorably converts AA into noninterfering species during the incubation period. The detection limit for VV in commercial 50% mandarin juice (AA concentration = 0.7 mM) is 4 × 10(3) plaque-forming unit (PFU) per mL. The new EC redox cycling scheme is promising for sensitive detection of proteins, viruses, and bacteria in solutions containing high concentrations of AA.

Low-interference washing-free electrochemical immunosensor using glycerol-3-phosphate dehydrogenase as an enzyme label.

In washing-free electrochemical detection, various redox and reactive species cause significant interference. To minimize this interference, we report a washing-free electrochemical immunosensor using flavin adenine dinucleotide (FAD)-dependent glycerol-3-phosphate dehydrogenase (GPDH) and glycerol-3-phosphate (GP) as an enzyme label and its substrate, respectively, because the reaction of FAD-dependent dehydrogenases with dissolved O2 is slow and the level of GP preexisting in blood is low (<0.1 mM). A combination of a low electrocatalytic indium-tin oxide (ITO) electrode and fast electron-mediating Ru(NH3)6(3+) is employed to obtain a high signal-to-background ratio via proximity-dependent electron mediation of Ru(NH3)6(3+) between the ITO electrode and the GPDH label. Electrochemical oxidation of GPDH-generated Ru(NH3)6(2+) is performed at 0.05 V vs Ag/AgCl, at which point the electrochemical interference is very low. When a washing-free immunosensor is applied to cardiac troponin I detection in human serum, the calculated detection limit is approximately 10 pg/mL, indicating that the immunosensor is very sensitive in spite of the use of washing-free detection with a short detection period (10 min for incubation and 100 s for electrochemical measurement). The low-interference washing-free electrochemical immunosensor shows good promise for fast and simple point-of-care testing.

Washing-free heterogeneous immunosensor using proximity-dependent electron mediation between an enzyme label and an electrode.

Washing processes, essential in most heterogeneous labeled assays, have been a big hurdle in simplifying the detection procedure and reducing assay time. Nevertheless, less attention has been paid to washing-free heterogeneous labeled assays. We report a purely washing-free immunosensor that allows fast, sensitive, and single-step detection of prostate-specific antigen in serum with low interference. Proximity-dependent electron mediation of ferrocenemethanol (Fc) between an indium-tin oxide (ITO) electrode and a glucose-oxidase (GOx) label allows us to discriminate between a bound and an unbound label: a bound label offers faster electron mediation than an unbound one. The electrooxidation of Fc at a low applied potential (0.13 V vs Ag/AgCl) and a low electrocatalytic ITO electrode and the oxidation of l-ascorbic acid by l-ascorbate oxidase minimize the effect of the interfering species. With a high concentration of glucose (200 mM), the signal and background levels are hardly dependent on the glucose-concentration variation in the sample. The washing-free immunosensor can detect a concentration of ca. 1 pg/mL for mouse IgG in phosphate-buffered saline and a concentration of ca. 10 pg/mL for prostate-specific antigen spiked in female serum after an incubation period of 10 min. The concentrations measured with actual clinical serum samples are in good agreement with the concentrations measured with a commercial instrument, which renders the washing-free heterogeneous immunosensor appealing for practical use.

Facile decrease in the electron-transfer rate and surface roughness of gold by ultrasonic treatment.

This communication reports that the electron-transfer rate and surface roughness of Au electrodes can be decreased simply by ultrasonic treatment. It seems that the hydroxyl radical generated during ultrasonic treatment plays an important role, as in the case of treatment with Fenton's reagent.