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The WHO estimates that 8–10% of couples are facing fertility problems, often due to inaccuracy in predicting the female's ovulation period controlled by four key hormones. The quantification and monitoring of such key hormones are crucial for the early identification of infertility, but also in improving therapeutic management associated with hormonal imbalance. In this review, we extensively summarize and discuss: i) drawbacks of laboratory methods for fertility testing (costly, invasive, complex) and commercially available point-of-care tests (measuring only one/two of the four key hormones), ii) the understanding of different biosensors for fertility monitoring, and iii) an in-depth classification and overview of aptamer-based sensing of the hormones of interest. This review provides insights on hormone detection strategies for fertility, with a focus on the classification of the current ‘aptasensing’ strategies, aiming to assist as a basic guide for the development of accurate fertility window monitoring tools based on aptamers.

Strains of Penicillium spp. are used for fungi-ripened cheeses and Aspergillus spp. routinely contaminate maize and other crops. Some of these strains can produce toxic secondary metabolites (mycotoxins), including the neurotoxin α-cyclopiazonic acid (CPA). In this work, we developed a homogeneous upconversion-resonance energy transfer (UC-RET) immunoassay for the detection of CPA using a novel epitope mimicking peptide, or mimotope, selected by phage display. CPA-specific antibody was used to isolate mimotopes from a cyclic 7-mer peptide library in consecutive selection rounds. Enrichment of antibody binding phages was achieved, and the analysis of individual phage clones revealed four different mimotope peptide sequences. The mimotope sequence, ACNWWDLTLC, performed best in phage-based immunoassays, surface plasmon resonance binding analyses, and UC-RET-based immunoassays. To develop a homogeneous assay, upconversion nanoparticles (UCNP, type NaYF4:Yb3+, Er3+) were used as energy donors and coated with streptavidin to anchor the synthetic biotinylated mimotope. Alexa Fluor 555, used as an energy acceptor, was conjugated to the anti-CPA antibody fragment. The homogeneous single-step immunoassay could detect CPA in just 5 min and enabled a limit of detection (LOD) of 30 pg mL-1 (1.5 μg kg-1) and an IC50 value of 0.36 ng mL-1. No significant cross-reactivity was observed with other co-produced mycotoxins. Finally, we applied the novel method for the detection of CPA in spiked maize samples using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) as a reference method. This work has been funded by the Ministry of Science, Innovation and Universities (MSIU) (RTI2018-096410-B-C21, PID2021-127457OB-C21 and PID2019-105237 GB-I00). FP acknowledges the MSIU for an FPU contract. Sí

Antibody profiling is a fundamental component of understanding the humoral response in a wide range of disease areas. Most currently used approaches operate by capturing antibodies onto functionalised surfaces. Such measurements of surface binding are governed by an overall antibody titre, while the two fundamental molecular parameters, antibody affinity and antibody concentration, are challenging to determine individually from such approaches. Here, by applying microfluidic diffusional sizing (MDS), we show how we can overcome this challenge and demonstrate reliable quantification of alloantibody binding affinity and concentration of alloantibodies binding to Human Leukocyte Antigens (HLA), an extensively used clinical biomarker in organ transplantation, both in buffer and in crude human serum. Capitalising on the ability to vary both serum and HLA concentrations during MDS, we show that both affinity and concentration of HLA-specific antibodies can be determined directly in serum when neither of these parameters is known. Finally, we provide proof of principle in clinical transplant patient sera that our assay enables differentiation of alloantibody reactivity against HLA proteins of highly similar structure, providing information not attainable through currently available techniques. These results outline a path towards detection and in-depth profiling of humoral immunity and may enable further insights into the clinical relevance of antibody reactivity in clinical transplantation and beyond.

The current work, designed for the photoelectrochemical detection of DNA, evaluates light-responsive DNA probes carrying molecular photosensitizers generating singlet oxygen (1O2). We take advantage of their chromophore’s ability to produce 1O2 upon photoexcitation and subsequent photocurrent response. Type I, fluorescent and type II photosensitizers were studied using diode lasers at 406 nm blue, 532 nm green and 659 nm red lasers in the presensce and absence of a redox reporter, hydroquinone (HQ). Only type II photosensitizers (producing 1O2) resulted in a noticeable photocurrent in 1–4 nA range upon illumination, in particular, dissolved DNA probes labeled with chlorin e6 and erythrosine were found to give a well-detectable photocurrent response in the presence of HQ. Whereas, Type I photosensitizers and fluorescent chromophores generate negligible photocurrents (

With the increased demand for beef in emerging markets, the development of quality-control diagnostics that are fast, cheap and easy to handle is essential. Especially where beef must be free from pork residues, due to religious, cultural or allergic reasons, the availability of such diagnostic tools is crucial. In this work, we report a label-free impedimetric genosensor for the sensitive detection of pork residues in meat, by leveraging the biosensing capabilities of graphene acid - a densely and selectively functionalized graphene derivative. A single stranded DNA probe, specific for the pork mitochondrial genome, was immobilized onto carbon screen-printed electrodes modified with graphene acid. It was demonstrated that graphene acid improved the charge transport properties of the electrode, following a simple and rapid electrode modification and detection protocol. Using non-faradaic electrochemical impedance spectroscopy, which does not require any electrochemical indicators or redox pairs, the detection of pork residues in beef was achieved in less than 45 min (including sample preparation), with a limit of detection of 9% w/w pork content in beef samples. Importantly, the sample did not need to be purified or amplified, and the biosensor retained its performance properties unchanged for at least 4 weeks. This set of features places the present pork DNA sensor among the most attractive for further development and commercialization. Furthermore, it paves the way for the development of sensitive and selective point-of-need sensing devices for label-free, fast, simple and reliable monitoring of meat purity., We acknowledge funding from the European Union Horizon2020 Programme under Grant No. 881603 (Graphene Flagship Core 3). This article reflects only the author's view, and the European Commission is not responsible for any use that may be made of the information it contains. ICN2 is funded by the CERCA programme, Generalitat de Catalunya. The ICN2 is supported by the Severo Ochoa Centres of Excellence programme, funded by the Spanish Research Agency (AEI, grant no. SEV-2017-0706). J. M. R. Flauzino is grateful for the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior scholarship (CAPES-PRINT, Brazil, Grant number: 88887.371591/2019–00). D. Panáček acknowledges the Internal Student Grant Agency of the Palacký University in Olomouc, (IGA_PrF_2021_031). A. G. Brito-Madurro acknowledges the funding from Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (310782/2018–0). J. M. Madurro acknowledges the funding from Fundação de Amparo à Pesquisa do Estado de Minas Gerais – FAPEMIG (CEX-APQ-02902-17) and Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (311737/2018–8). A. Bakandritsos acknowledges the funding from the Czech Science Foundation, (project GA CR – EXPRO, 19–27454X). M. Otyepka acknowledges the ERC grand 2D-CHEM (No 683024 from H202). The work was supported also by the ERDF/ESF project “Nano4Future” (No.CZ.02.1.01/0.0/0.0/16_019/0000754).

The detection of extracellular pH at single cell resolution is challenging and requires advanced sensibility. Sensing pH at high spatial and temporal resolution might provide crucial information in understanding the role of pH and its fluctuations in a wide range of physio-pathological cellular processes, including cancer. Here, a method to embed silica-based fluorescent pH sensors into alginate-based three-dimensional (3D) microgels tumour models, coupled with a computational method for fine data analysis, is presented. By means of confocal laser scanning microscopy, live-cell time-lapse imaging of 3D alginate microgels was performed and the extracellular pH metabolic variations were monitored in both in vitro 3D mono- and 3D co-cultures of tumour and stromal pancreatic cells. The results show that the extracellular pH is cell line-specific and time-dependent. Moreover, differences in pH were also detected between 3D monocultures versus 3D co-cultures, thus suggesting the existence of a metabolic crosstalk between tumour and stromal cells. In conclusion, the system has the potential to image multiple live cell types in a 3D environment and to decipher in real-time their pH metabolic interplay under controlled experimental conditions, thus being also a suitable platform for drug screening and personalized medicine.

Site-selective drug effects on the ion-channel activities of γ-aminobutyric acid type A (GABA

The organized self-assembly of conductive biological structures holds promise for creating new bioelectronic devices. In particular, Geobacter sulfurreducens type IVa pili have proven to be a versatile material for fabricating protein nanowire-based devices. To scale the production of conductive pili, we designed a strain of Shewanella oneidensis that heterologously expressed abundant, conductive Geobacter pili when grown aerobically in liquid culture. S. oneidensis expressing a cysteine-modified pilin, designed to enhance the capability to bind to gold, generated conductive pili that self-assembled into biohybrid filaments in the presence of gold nanoparticles. Elemental composition analysis confirmed the filament-metal interactions within the structures, which were several orders of magnitude larger than previously described metal:organic filaments. The results demonstrate that the S. oneidensis chassis significantly advances the possibilities for facile conductive protein nanowire design and fabrication.

After discovery of electron transfer from bacteria, most bacteria known to be electrochemically active are utilized as a self-regenerable catalyst at the anode of microbial fuel cells (MFCs). However, the reverse phenomenon, cathodic catalysts is not so widely researched. This present study demonstrated that E. coli O157:H7 was electrochemically active, and it was able to catalyze oxygen reduction at the cathode of bipolar electrode (BPE). Applying a constant potential to the BPE, E. coli O157:H7 can catalyze electrochemical reduction of O

Identifying chemical odors rapidly and accurately is critical in a variety of fields. Due to the limited human sense of smell, much effort has been dedicated to the development of electronic sensing devices. Despite some recent progress, such devices are still no match for the capabilities of biological (animal) olfactory sensors, which are light, robust, versatile, and sensitive. Consequently, scientists are turning to a new approach: Bio-Hybrid sensors. These sensors combine animal biological sensors with electronic components to achieve maximum detection and classification while conveying a comprehensible signal to the end user. In this work, we created a bio-hybrid odor discriminator utilizing the desert locust's primary olfactory apparatus - its antennae, together with simple electroantennogram technology and artificial intelligence tools for signal analysis. Our discriminator is able to differentiate between at least eight pure odors and two mixtures of different odorants, independently of odorant concentration. With four orders of magnitude higher sensitivity than gas chromatography-mass spectrometry, it is able to detect the presence of less than 1 ng of volatile compounds and, compared to other bio-hybrid sensors available today, it can be easily operated by an unskilled individual. This study thus opens up the future for robust and simple bio-hybrid robotic sensing devices that can be widely deployed.

Exosomal microRNAs (miRNAs) are emerging as attractive non-invasive and reliable biomarkers for disease diagnosis. In situ exosomal miRNA detection can avoid laborious and time-consuming exosome lysis, RNA extraction and effectively improve the accuracy. However, in situ exosomal miRNA detection is hampered by the low abundance of the targets and low permeability of the probes. Herein, an in situ exosomal miRNA sensing biochip based on multi-branched localized catalytic hairpin assembly (MLCHA) and photonic crystals (PCs) was proposed. The MLCHA probes could penetrate into the exosomes nondestructively due to its rigidity and generate amplified fluorescence signal upon recognizing the target miRNA. And then, the fluorescence signal was further enhanced by PCs to improve the sensitivity. The developed biosensor can not only detect exosomal miRNA in a concentration-dependent manner but also distinguish samples from cancer state and healthy state, which is potential for non-invasive clinical diagnostics.

Quick and easy monitoring approaches are needed to assess sources, clinical relevance, and incidences of antibiotic resistance in environmental bacterial communities. In the present work, antibiotic-resistant bacterial contamination detection (ABCD) kits were developed for the same. The method was standardized with strains of Escherichia coli TS7, Staphylococcus arlettae HWI8, Enterococcus faecalis HWI19, and Aerococcus viridans HWII16 with known antibiogram using six clinically important antibiotics. The method was verified with different water sources having different physicochemical parameters successfully. Only 1 ml of sample water is required to be mixed with an optimized concentration of the antibiotic solution and incubated for 6h; subsequently, a color change to pink may be observed within a specified amount of time upon the addition of the bacterial detection PVDF membrane to monitor the presence of resistant bacteria. There is no color change in the case of antibiotic-susceptible bacterial communities or the absence of a resistant community. Moreover, the time taken for color change is inversely related to the magnitude of the antibiotic-resistant communities in terms of enumeration. Up to our understanding, this is the 1st report which can determine an antibiotic-resistance profile of any water source by observing only color change within a maximum of 7 h (6 h for co-culture of bacteria and antibiotics + 1 h for color change detection) time without the aid of any microbiology laboratory or skilled manpower.

Whole-cell biosensors based on transcriptional regulators are powerful tools for rapid measurement, high-throughput screening, dynamic metabolic regulation, etc. To optimize the biosensing performance of transcriptional regulator, its effector-binding domain is commonly engineered. However, this strategy is encumbered by the limitation of diversifying such a large domain and the risk of affecting effector specificity. Molecular dynamics simulation of effector binding of LysG (an LysR-type transcriptional regulator, LTTR) suggests the crucial role of the short linker helix (LH) connecting effector- and DNA-binding domains in protein conformational change. Directed evolution of LH efficiently produced LysG variants with extended operational range and unaltered effector specificity. The whole-cell biosensor based on the best LysG

Cancer spheroids, which mimic distinct cell-to-cell and cell-extracellular matrix interactions of solid tumors in vitro, have emerged as a promising tumor model for drug screening. However, owing to the unique characteristics of spheroids composed of three-dimensionally densely-packed cells, the precise characterizations of cell viability and function with conventional colorimetric assays are challenging. Herein, we report gold nanostructure-integrated conductive microwell arrays (GONIMA) that enable both highly efficient uniform cancer spheroid formation and precise electrochemical detection of cell viability. A nanostructured gold on indium tin oxide (ITO) substrate facilitated the initial cell aggregation and further 3D cell growth, while the non-cytophilic polymer microwell arrays restricted the size and shape of the spheroids. As a result, approximately 150 human glioblastoma spheroids were formed on a chip area of 1.13 cm

Measuring glucose together with glycated hemoglobin (HbA1c) could provide short-term and long-term information of blood glycemic condition after a single treatment. However, it has been a challenge to quantify glucose and HbA1c from single sample drop and strip of electrodes with suitable sensitivity, selectivity, and efficiency. In this paper, we demonstrated a label freesingle sample drop electrochemical detection method of glucose and HbA1c by modifying carbon electrodes. Glucose oxidase and capture antibodies (C-Ab) against HbA1c were immobilized onto the first working electrode (WE1) and the second working electrode (WE2) of dual working screen-printed carbon electrodes (DWSPCEs), respectively. WE2 was modified with Gold Nano Flower. After that, 3-mercaptopropionic acid was coated as a linker. Finally, C-Ab was bonded by the linker. The relationship between gold surface area and concentration of HgAuCl

The devastating effects of global climate change on crop production and exponential population growth pose a major challenge to agricultural yields. To cope with this problem, crop performance monitoring is becoming increasingly necessary. In this scenario, the use of sensors and biosensors capable of detecting changes in plant fitness and predicting the evolution of their morphology and physiology has proven to be a useful strategy to increase crop yields. Flexible sensors and nanomaterials have inspired the emerging fields of wearable and on-plant portable devices that provide continuous and accurate long-term sensing of morphological, physiological, biochemical, and environmental parameters. This review provides an overview of novel plant sensing technologies by discussing wearable and integrated devices proposed for engineering plant and monitoring its morphological traits and physiological processes, as well as plant-environment interactions. For each application scenario, the state-of-the-art sensing solutions are grouped according to the plant organ on which they have been installed highlighting their main technological advantages and features. Finally, future opportunities, challenges and perspectives are discussed. We anticipate that the application of this technology in agriculture will provide more accurate measurements for farmers and plant scientists with the ability to track crop performance in real time. All of this information will be essential to enable rapid optimization of plants development through tailored treatments that improve overall plant health even under stressful conditions, with the ultimate goal of increasing crop productivity in a more sustainable manner.

Glucose detection is vital in the food industry for safety and quality management. As a healthy ingredient, the flavor of honey is frequently impacted by the crystallization of glucose. Therefore, determining the glucose level can offer precise reference data for the manufacture of honey. Various approaches have been tried, and the enzyme-based electrochemical analytical method is one of the most important and widely used strategies. However, there are still challenges for most electrochemical methods to achieve stable detection resistant to temperature variation due to the easy inactivation of the enzyme, the poor anti-interference capacity of the detection techniques and other influences from the external environment. Herein, a hydrogel-based electrochemical biosensor is proposed to stably detect glucose even at wide ranges of temperatures via electrochemical impedance spectroscopic (EIS) measurement. The key factor for stable detection relies on the metal-organic framework nanoparticles' protective layer to guarantee the robustness of glucose oxidase (GOx), thereby achieving stable and specific detection for glucose. Moreover, a cascade reaction-induced hydrogel formation in a 3D structure can be used as an impedance readout, which not only amplifies but also further stabilizes the GOx-induced response. The prepared hydrogel-based electrochemical biosensor showed a linear response to the glucose concentration in the range of 0.75-4 mg/mL. Furthermore, the biosensor has excellent anti-interference and temperature stability. High performance liquid chromatography analysis also validated the accuracy of this biosensor in detecting glucose in the honey sample.

For point-of-care testing (POCT), coupling isothermal nucleic acid amplification schemes (e.g., recombinase polymerase amplification, RPA) with lateral flow assay (LFA) readout is an ideal platform, since such integration offers both high sensitivity and deployability. However, isothermal schemes typically suffers from non-specific amplification, which is difficult to be differentiated by LFA and thus results in false-positives. Here, we proposed an accurate POCT platform by specific recognition of target amplicons with peptide nucleic acid (PNA, assisted by T7 Exonuclease), which could be directly plugged into the existing RPA kits and commercial LFA test strips. With SARS-CoV-2 as the model, the proposed method (RPA-TeaPNA-LFA) efficiently eliminated the false-positives, exhibiting a lowest detection concentration of 6.7 copies/μL of RNA and 90 copies/μL of virus. Using dual-gene (orf1ab and N genes of SARS-CoV-2) as the targets, RPA-TeaPNA-LFA offered a high specificity (100%) and sensitivity (RT-PCR Ct 31, 100%; Ct 40, 71.4%), and is valuable for on-site screening or self-testing during isolation. In addition, the dual test lines in the test strips were successfully explored for simultaneous detection of SARS-CoV-2 and H1N1, showing great potential in response to future pathogen-based pandemics.

Classification and sorting of cells using image-activated cell sorting (IACS) systems can bring significant insight to biomedical sciences. Incorporating deep learning algorithms into IACS enables cell classification and isolation based on complex and human-vision uninterpretable morphological features within a heterogeneous cell population. However, the limited capabilities and complicated implementation of deep learning-assisted IACS systems reported to date hinder the adoption of the systems for a wide range of biomedical research. Here, we present image-activated cell sorting by applying fast deep learning algorithms to conduct cell sorting without labeling. The overall sorting latency, including signal processing and AI inferencing, is less than 3 ms, and the training time for the deep learning model is less than 30 min with a training dataset of 20,000 images. Both values set the record for IACS with sorting by AI inference. . We demonstrated our system performance through a 2-part polystyrene beads sorting experiment with 96.6% sorting purity, and a 3-part human leukocytes sorting experiment with 89.05% sorting purity for monocytes, 92.00% sorting purity for lymphocytes, and 98.24% sorting purity for granulocytes. The above performance was achieved with simple hardware containing only 1 FPGA, 1 PC and GPU, as a result of an optimized custom CNN UNet and efficient use of computing power. The system provides a compact, sterile, low-cost, label-free, and low-latency cell sorting solution based on real-time AI inferencing and fast training of the deep learning model.

A novel electrochemical biosensor was developed to monitor fibroblast cells stress levels for the first time in situ under external stimuli based on the recognition of superoxide anion released upon cell damage. The biosensor comprised metallized polycaprolactone electrospun fibers covered with zinc oxide for improved cell adhesion and signal transduction, whilst stable bioconjugates of mercaptobenzoic acid-functionalized gold nanoparticles/superoxide dismutase were employed as recognition bioelements. Biosensors were first tested and optimized for in situ generated superoxide detection by fixed potential amperometry at +0.3 V, with minimal interferences from electroactive species in cell culture media. L929 fibroblast cells were then implanted on the optimized biosensor surface and the biosensor morphologically characterized by scanning electron microscopy (SEM) and fluorescence microscopy, which illustrated the network-type pattern of fibroblasts adjacent to the fiber scaffold. Fibroblast stress was induced by zymosan and monitored at the cells integrated biosensor using fixed potential amperometry (CA) with a sensitivity of 26 nA cm

A photoelectrochemical (PEC) immunosensor based on MIL-101(Cr) and CdTe-QDs composites (MC) was successfully synthesized to assay the carcinoembryonic antigen (CEA) in human serum and urine samples. This novel method contained three key aspects: 1), the polarity of the photocurrent based on MIL-101(Cr) itself could be altered by applying a different voltage to detect the cathode and anode photocurrent. 2), the introduction of cadmium telluride quantum dots (CdTe-QDs) greatly improved the efficiency of light utilization. 3), the photogenerated electron-hole-pairs were suppressed and their separation efficiency was improved by effective matching of energy level between MIL-101(Cr) and CdTe-QDs. Due to the inherent insulating properties of the biological matrix, the transfer of photogenerated electrons was hindered, leading to a decreased photocurrent signal. Under the optimal condition, the anodic and cathodic detection limit of the PEC immunosensor for CEA was 0.00018 ng mL

Heart disease is the leading cause of death worldwide and imposes a significant burden on healthcare systems globally. A major hurdle to the development of more effective therapeutics is the reliance on animal models that fail to faithfully recapitulate human pathophysiology. The predictivity of in vitro models that lack the complexity of in vivo tissue remain poor as well. To combat these issues, researchers are developing organ-on-a-chip models of the heart that leverage the use of human induced pluripotent stem cell-derived cardiomyocytes in combination with novel platforms engineered to better recapitulate tissue- and organ-level physiology. The integration of novel biosensors into these platforms is also a critical step in the development of these models, as they allow for increased throughput, real-time and longitudinal phenotypic assessment, and improved efficiency during preclinical disease modeling and drug screening studies. These platforms hold great promise for both improving our understanding of heart disease as well as for screening potential therapeutics based on clinically relevant endpoints with better predictivity of clinical outcomes. In this review, we describe state-of-the-art heart-on-a-chip platforms, the integration of novel biosensors into these models for real-time and continual monitoring of tissue-level physiology, as well as their use for modeling heart disease and drug screening applications. We also discuss future perspectives and further advances required to enable clinical trials-on-a-chip and next-generation precision medicine platforms.

In this work, a portable electrochemiluminescence (ECL) detection system based on silicon photomultiplier (SiPM) single photon detector was proposed for the detection of ECL signals on a screen-printed electrode (SPE). This instrument innovatively used SiPM single photon detector to detect the ECL signal, which solved friability and bloat caused by the high operating voltage and the limitation of detection components in the traditional ECL detection instrument. This detection instrument showed excellent electrochemical and ECL detection performance. On this basis, an aptasensor based on silver (core)-gold (shell) bimetallic nanoparticles (Ag@AuNPs) was constructed for the detection of tetracycline (TET) in milk on SPE. Here, Ag@AuNPs had a significant effect in enhancing luminol ECL signal and immobilizing aptamer. The concentration of TET was detected according to the changes of the ECL signal intensity of the detection instrument. This instrument exhibited an excellent linearity ranging from 0.01 ng/mL to 1,000 ng/mL for the detection of TET, and a limit of detection (LOD) was 0.0053 ng/mL. The developed portable ECL detection instrument provides a new platform for the detection of small molecule contaminants.

The study of subcellular targets and biochemical processes within a living cell is valuable for biological and medical research. Secretory vesicles, one such important intracellular target, are nanoscale lipid structures that are capable of storage, transport, and secretion of, for example, neurotransmitters, hormones, proteins or waste products. Vesicles play an essential role in intercellular communication systems, as they facilitate the release of chemical messaging agents. If deregulated, these communication processes can be a central part in the pathogenesis of some neurodegenerative diseases or diabetes. Generally, due to their nanometer size and intracellular location, the analysis of single vesicles and their content is a great challenge. It requires sensitive techniques, micro/nanoscale tools and sensitive instruments with extreme spatio-temporal resolution. This review focuses on electrochemical sensors to study the biochemistry and quantification of messenger molecules and other species (e.g., reactive oxygen and nitrogen species) stored in organelles, providing new trends and developments in this field. Furthermore, we review the effect of the chemical environment of single cells (e.g., treatment with chemicals, drugs, lipids, and ions) on regulation of the physical and chemical properties of vesicles. Finally, unsolved challenges of and perspectives on vesicle electroanalysis are discussed.

Based on the excellent properties, photothermal nanomaterials have been widely used in many fields, such as trace substance detection, optical imaging, medical diagnosis and treatment. Different from colorimetric, fluorescent and electrochemical methods which often suffer from the problems of high background interference, operation procedure dependence and high cost, photothermal sensing can effectively break the limitations of the above methods and become an alternative strategy due to its high resolution and spatial controllability. Especially in recent years, the continuous development of new materials and the introduction of portable, low-cost and precision photothermal measuring equipment can significantly improve the application potential of photothermal detection. Here, we conducted comprehensive analyses on those studies. This review summarized the progress of photothermal detection over recent years, including the main types of photothermal nanomaterials and the generation principle of the photothermal effect, the relevant matching thermometry tools, and the applications in the designing of sensing strategies. It aims to provide a reference for researchers to synthesize nanomaterials with stable and high photothermal conversion efficiency and develop more effective and sensitive detection methods.

MicroRNAs (miRNAs) play an important role in post-transcriptional regulation of gene expression. However, methods to accurately detect miRNA activity in living cells are still limited. Here we developed a DNA nanomachine initiated by a miRNA-induced silencing complex (miRISC) for imaging miRNA activity in living cells. miRISC-mediated RNA cleavage reaction activated the DNA nanomachine by the specific cleavage of an RNA strand on the machine, resulting in autonomous movement of the walking leg around the AuNP surface with the release of a large number of fluorescently labeled DNA strands. The DNA nanomachine was successfully applied to detect miR-21 activity in three cell lines with different miR-21 expression profiles. We also demonstrated that terminal uridylyltransferase Tut4 knockdown by siRNA significantly increased the activity of let-7b miRNA, which further verifies the versatility of our DNA nanomachine. This new nanomachine has distinct advantages compared with reported methods for detecting miRNA activity, including simple operating procedures, short analysis time and sensitive signal output. Collectively, this work not only expands the application of the DNA nanomachine in the detection of miRNA activity, but also provides a promising tool for basic research in cell biology and development of clinical biomedicine.

Analysis of cytokines levels in human serum is critical as it can be a "symptom diagnostic biomarker" in COVID-19, giving real-time information about human health status. Here, we present the construction and performance of a low-price immunosensor (∼US$0.428 per test) based on microfluidic paper-based system to detect cytokine for predicting the health status of COVID-19 patients. Interleukin-6 (IL-6) was selected as the detection model for the close relationship between IL-6 and COVID-19. The assay, which we integrated into foldable paper system, leverages the magnetic immunoassay, the streptavidin-horseradish peroxidase (HRP) associated with tetramethyl benzidine/hydrogen peroxide (TMB/H

We propose a label-free biosensor based on a porous silicon resonant microcavity and localized surface plasmon resonance. The biosensor detects SARS-CoV-2 antigen based on engineered trimeric angiotensin converting enzyme-2 binding protein, which is conserved across different variants. Robotic arms run the detection process including sample loading, incubation, sensor surface rinsing, and optical measurements using a portable spectrometer. Both the biosensor and the optical measurement system are readily scalable to accommodate testing a wide range of sample numbers. The limit of detection is 100 TCID

The interest in ketone bodies (KBs) has intensified recently as they play significant roles in healthcare, nutrition, and wellness applications. We present a disposable electrochemical sensing strip for rapid decentralized detection of β-hydroxybutyrate (HB), one of the dominant physiological KBs, in saliva. The new salivary enzymatic HB sensor strip relies on a gold-coated screen-printed carbon electrode modified with a reagent layer containing toluidine blue O (TBO mediator), β-hydroxybutyrate dehydrogenase (HBD enzyme), and the HBD cofactor nicotinamide adenine dinucleotide (NAD

Carbapenem-resistant organisms (CROs) are characterized by high drug resistance, rapid transmission, and high lethality. Therefore, rapid detection for CROs is essential for appropriate applying antibiotics and implementing quarantine. Droplet digital chromogenic assays (DDCA) have been accepted as an effective means for rapid microbial detection as the small droplet volumes facilitate a significant enhancement in the local concentration of chromogenic factors and, therefore, reduce the required time of the test. Nevertheless, as their dependence on the time-consuming isolation culture, the DDCA is still associated with a long turnaround time. To overcome this limitation, we develop here a microfluidic chip-based CRO phenotypic identification method that integrates cascade filtration (CF) with DDCA (CF-DDCA). After a body fluid sample is introduced to the microfluidic chip, particles with sizes5 μm are removed out by the primary filter, and Gram (+) cocci with sizes1 μm removed out by the secondary filter so that only Gram (-) bacilli with sizes between 1.5 and 5 μm are selectively retained. The purified Gram (-) bacilli, along with chromogenic reagents and carbapenem antibiotics, are then subjected to the DDCA. We demonstrate that the CF can remove 99.9% of the interfering microorganisms and thus eliminates the isolating culture. Benefited from the isolating culture-free DDCA, phenotypic identification of CROs can be achieved within 3.5 h. Clinical urine sample testing shows that the sensitivity and specificity of the CF-DDCA for CRO identification are all 100%, and the total coincidence rate between CF-DDCA and the conventional assay is also 100%.

Paralytic shellfish toxins (e.g., saxitoxin, STX; gonyautoxin-2, GTX-2) and tetrodotoxin (TTX) are highly toxic and widely distributed ion channel marine toxins which specifically block the voltage-dependent sodium channels (VDSCs), causing great harm to human health. It is urgent to exploit new detection methods with high specificity and high efficiency. Here, a portable high-throughput cardiomyocyte-based potential biosensor was established with cardiomyocytes, a 16-well microelectrodes (MEs) sensor and a robust 32-channel recording system, which presented high-quality and high-consistency extracellular field potential (EFP) signals in each well with a long duration of 80 h. The feature parameters, including firing rate (FR), spike amplitude (SA), spike slope (SS), spike duration (SD) and field potential duration (FPD), were extracted from EFP to quantitatively assess the toxic effects of these ion channel toxins. Importantly, the biosensor showed temporal specificity and parametric selectivity under toxin treatments, and FR, SS and SD were the optimal parameters to STX, TTX and GTX-2, respectively. This biosensor can rapidly detect 0.29 ng/mL STX, 0.30 ng/mL TTX and 0.16 ng/mL GTX-2 within 5 min, 10 min and 15 min, respectively. Thus, our novel multi-well cardiomyocyte-based biosensor will be a promising tool for high-effective detection of ion channel toxins.

An electro-plasmonic biosensor is used to attract proteins and cells on the surface of a fiber optic probe by controlled biomolecular migration. Concentrating targets on a high performance plasmon-assisted fiber grating sensor leads to a drastic enhancement of the limit of detection. This architecture relies on a biofunctionalized gold coated tilted fiber Bragg grating (TFBG) that operates as a working electrode to enable electrophoresis in the probed medium. The applied electric field triggers the attraction of proteins over a distance of almost 250 μm from the sensor surface, which is more than two orders of magnitude larger than the intrinsic penetration depth of the plasmon wave. Quantitative determination of target analytes was performed by cyclic voltammetry measurements using the gold coated fiber as an electrode, simultaneously with optical transmission measurements of the underlying fiber grating. In our work, these electro-plasmonic optrodes were used against a clinically-relevant biomarker in breast cancer diagnosis, namely HER2 (Human Epidermal Growth Factor Receptor-2). In vitro assays confirm that their limit of detection lies in the subpicomolar range for proteins, which is beyond reach of similar sensors without voltammetry. The improved detection limit is further facilitated by an improvement of the signal-to-noise ratio of the read-out process. Whole cell capture is finally demonstrated by the same micro-system.

Extensively industrial applications and ever-accelerated anthropogenic activities have resulted in the dramatic accumulation of Sb

Early finding of pathogens is significant to avoid foodborne diseases. Here, a novel lab-in-centrifugal-tube colorimetric biosensor was reported for Salmonella typhimurium detection using immune nickel nanowires (NNWs) to form capture nets for specific bacterial separation, gold@platinum nanozymes (GPNs) to mark target bacteria for effective signal amplification, and a smartphone App to analyze color change for quantitative bacterial determination. A 3D-printed cylindrical magnetic separator with air pressure self-regulating structure and NNW capture nets was elaboratively constructed and assembled inside the disposable centrifuge tube to simply perform the bacterial separation, label, wash, coloration and detection. Under optimal conditions, Salmonella typhimurium could be quantitatively detected in 2 h with a low detection limit of 21 CFU/mL. The recovery of target bacteria in spiked pork samples ranged from 87.0% to 97.6% with the averaged recovery of 93.9%. This biosensor was Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users (ASSURED), and had shown the potential for point-of-care testing of foodborne pathogens to ensure food safety.

Although serum prostate specific antigen (PSA) testing could decrease the morality of prostate cancer (PCa), its low specificity usually led to misdiagnosis due to prostatitis or benign prostatic hyperplasia (BPH). Prostate cancer antigen 3 (PCA3) as an alternative prostate tumor-specificity biomarker could be used to increase the specificity of PCa diagnosis, however, it usually required sophisticated operation and expensive equipment for routine detection. Herein, we constructed an early detection platform for prostate cancer with reverse transcriptase-recombinase aided amplification (RT-RAA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 based nucleic acid test strip. The amplicons of PCA3 and kallikrein related peptidase 3 (KLK3) gene, which amplified simultaneously by single-amplification unit of RT-RAA were specifically recognized by Cas9-sgRNA and visual on the nucleic acid test strip by naked eyes without instruments. Simultaneously detection of PCA3 and KLK3 gene could improve specificity and accuracy of the diagnosis but avoid mutual interference. In addition, the platform presented a detection limit of 500 fg/μL and 50 fg/μL in PCA3 and KLK3 gene, respectively. Furthermore, the analysis result of signal ratio of PCA3 to KLK3 gene of urine and peripheral blood specimens from 32 men with suspected prostate cancer on test strips illustrated that the area under the curve values of urine and peripheral blood specimens were 0.998 and 1.0 respectively. In summary, our study highlighted a facile strategy to design an accurate prostate cancer gene detection platform which had the potential to conduct prostate cancer early detection in the resource-limited or other point-of-care testing (POCT) environments.

Foodborne pathogens have raised significant concerns in human public health. Rapid, high-sensitive, low-cost, and easy-to-use testing methods for food safety are needed. In this study, we developed a finger-actuated microfluidic biosensor (FA-MB) for multiplexed detection of Bacillus cereus and other six common foodborne pathogens based on one-pot CRISPR/Cas12a combined with recombinase aided amplification (RAA). Wells for RAA and CRISPR/Cas12a were isolated to avoid interference, while finger-actuated one-way control valves were incorporated to fulfill the unidirectional flow of RAA products to the CRISPR/Cas12a reaction wells, realizing one-pot RAA-CRISPR/Cas12a assay. The final fluorescent signal was acquired and processed by a smartphone. Under selected experimental conditions, seven pathogenic bacteria could be tested in about 1 h with the limits of detection (LODs) below 500 CFU/mL. Recoveries ranged from 90% to 116% of the spiked samples were readily achieved. The proposed FA-MB is highly integrated and easy-to-use, and could be used for rapid, high-sensitive point of care (POC) testing without the external driving device, suitable for resource-constrained settings.

The thyroid gland, which regulates the metabolism of the human body, has a sophisticated feedback system that induces the secretion of thyroid-stimulating hormone (TSH) to regulate the levels of triiodothyronine (T3) and thyroxine (T4). In this study, a single-molecule fourplex nanoimmunosensor was developed for the simultaneous quantitative analysis of TSH, T3, and T4. The three thyroid hormones were detected with a high signal-to-noise ratio in an evanescent field using laser-induced total internal reflection fluorescence. Additionally, the use of gold nanoislands for the detection of molecular interactions between thyroid hormones and antibodies labeled with quantum dots minimized the background noise from the substrate compared with the use of microislands or microwells. The nanoimmunosensor exhibited excellent detection limits of 114-193 yM (yoctomolar = 10

Human epidermal growth factor receptor 2 (HER2) has been regarded as the considerable biomarker of breast and gastric cancer. Thus, precise detection of HER2 is of significance for the early diagnosis and treatment. Here, a photofuel cell-based self-powered biosensor (PFC-SPB) was constructed for the ultrasensitive HER2 detection, which was composed of a plasmonic gold nanoparticles (Au NPs)/organic semiconductor hybrid photoanode and a cathode with biosensing strategy of electrochemical sandwich structure. The localized surface plasmon resonance effect of Au NPs can obviously enhance the separation efficiency of photo-generated electron/hole pair, which was beneficial to the sensitivity and stability of PFC-SPB. Meanwhile, the cathodic sandwich structure not only was used for the target recognition, but also can guarantee the enrichment of electroactive molecules (molybdophosphate). Consequently, with the open circuit voltage (E

A novel colorimetric biosensor was explored for fast, sensitive, and on-site detection of Salmonella on a microfluidic chip employing immune gold@platinum nanoparticles (Au@Pt NPs) for specific bacterial labeling, a finger-driven mixer with two serial air chambers for efficient immunoreaction and a nuclear track membrane as specific-size microfilter for effective bacterial isolation from excessive immune Au@Pt NPs. First, the bacterial sample and immune Au@Pt NPs were pipetted into the mixing chamber and mixed sufficiently through repeated press-release actions on the small air chamber which could precisely control the air volume, leading to the formation of bacteria-immune Au@Pt NP conjugates. Then, the small and large air chambers were pressed simultaneously to push all the solution in the mixing chamber to flow through the microfilter for trap of the formed larger-size conjugates on the membrane and removal of the unbound smaller-size immune Au@Pt NPs. After the H

Yeast-based biosensors have great potential for various applications, although the present range of detectable chemicals is still very minimal. This work provides an enlargement of the knowledge on detectable chemicals and creates an additional basis for engineering modular yeast biosensors. Bacterial allosteric transcription factors, such as MarR and PdhR, were recruited to build transducer circuits in Saccharomyces cerevisiae. MarR-based biosensors were designed for the detection of aromatic permeant acids (benzoate and salicylate), whereas the PdhR-expressing yeast cells were engineered for responding to pyruvate. In general, all our engineered strains showed a fast response time and a strong fluorescent output signal to chemical concentrations ranging from 5 mM down to 2 fM. They exhibited versatile dynamic range and were capable of operating in a variety of complex media that might contain any of these compounds. A new milestone in biosensor design is the engineering of inter/intracellular metabolic biosensors that would allow real-time monitoring of either the metabolism of particular compounds, or the detection of their intermediate/end products. Our synthetic cells are applicable to different areas, from adequate real-time detection of aromatic permeant acids to regulation/monitoring of different hydrocarbon metabolisms. The new strains engineered in this study could be of great importance because of the ecological significance of aromatic permeant acids from their formations during either hydrocarbon degradation or metabolism of different chemicals to their involvement in different biological and non-biological systems.

Bipolar disorder is one of the severe mental diseases. Its high misdiagnosis rate and long-time delayed diagnosis are related to the fact that the diagnosis procedure is mainly conducted by doctors' subjective judgment. The diagnosis methods of bipolar disorder mainly include the International Classification of Diseases (ICD) or the Diagnostic and Statistical Manual of Mental Disorders (DSM) criteria and clinical guidelines based on clinical performance. To help psychiatrists make a more accurate diagnosis, in vitro diagnostic (IVD) techniques for bipolar disorder have been developed as the biomarkers research on bipolar disorder steadily increases. Here, we systematically review the recent studies in this area, summarizing the development of instant test products, potentially benefiting clinicians and their patients. The controversy over these biomarkers is discussed, pointing out that multilevel testing with more than one biomarker may provide better confidence in diagnoses. In some cases, more attention should be paid to the different reference values of some biomarkers in terms of age, gender, etc. The review on biomarkers for bipolar disorder may open new doors for the development of point-of-care testing (POCT) and instructing the RD of future products.

The assay performance of electrochemical DNA (E-DNA) sensors is deeply influenced by the state of DNA probes immobilized on electrode. Moreover, the immobilization procedures for DNA probes are tedious and vary according to the probes and analytes. In this work, we find that the adsorption layers of bovine serum albumin (BSA) on gold electrode (AuE) possess a size exclusion effect to distinguish between single-stranded (-ss) DNA probes and the DNA fragments generated from enzymatic digestion of ssDNA probes. In detail, the BSA layers act as a gatekeeper that hinders the adsorption of a ssDNA probe on AuE but permits the DNA fragments with much smaller sizes to pass through the adsorption layers and adsorb on AuE. This finding is developed into a novel E-DNA sensor for microRNA (miRNA) detection by coupling with duplex-specific nuclease (DSN)-assisted target recycling strategy. The ssDNA probe in solution phase is enzymatically digested during the DSN-assisted target recycling process initiated by target miRNA-21, generating plenty of DNA fragments. The adsorption of these DNA fragment on BSA/AuE is permitted, which arouses electrochemical signals after binding with [Ru(NH

There is a critical need for sensitive and rapid detection technologies utilizing molecular biotargets such as microRNAs (miRNAs), which regulate gene expression and are a promising class of diagnostic biomarkers for disease detection. Here, we present the development and fabrication of a highly reproducible and robust plasmonic bimetallic nanostar biosensing platform to detect miRNA targets using surfaced-enhanced Raman scattering (SERS)-based gene probes called the inverse Molecular Sentinel (iMS). We investigated and optimized the integration of iMS gene probes onto this SERS substrate, achieving ultra-sensitive detection with limits of detection of 6.8 and 16.7 zmol within the sensing region for two miRNA sequences of interest. Finally, we demonstrated the biomedical usefulness of this nanobiosensor platform with the multiplexed detection of upregulated miRNA targets, miR21 and miR221, from colorectal cancer patient plasma. The resulting SERS data are in excellent agreement with PCR data obtained from patient samples and can distinguish between healthy and cancerous patient samples. These results underline the potential of the iMS-integrated substrate nanobiosensing platform for rapid and sensitive diagnostics of cancer biomarkers for point-of-care applications.

Accurate and sensitive analysis of biomarkers is a promising way to provide comprehensive physio-pathological information that is significant for early diagnosis of certain diseases. miRNA is a type of noncoding small RNAs which are involved in the regulation of a number of cellular processes. It has been regarded as an important tumor biomarker. Herein, we have constructed a three-dimensional DNA layer on electrode interface and achieved ladder hybridization chain reaction strategy for the enrichment of electrochemical signals. In addition, duplex-specific nuclease catalyzed amplification is previously performed on magnetic nanocomposites, which further improves the sensitivity for the detection of target miRNA initiator. This approach shows great molecular recognition efficiency as well as cascade signal amplification. The analytical performances are superior. In addition, the identification of cancer cell types according to target biomarker information is achieved and the testing results in clinical serum samples further demonstrate its great potential utility for diagnosis.

Anode performance has been regarded as a crucial factor determining long-term stability and electricity generation of microbial fuel cells (MFCs), which restricts by the difficult extracellular electron transfer (EET) on the microbe/anode interface. Herein, inspired by biological enzyme systems, this study synthesized the biomimetic nanozymes with Fe-N-S-C active sites as the anode materials of MFCs, which was similar to the hemes of c-type cytochromes (c-Cyts) for boosting EET process. As excepted, an obviously faster start-up and a much higher power density were achieved by the MFCs equipped with Fe-N-S-C nanozymes (startup time, 3.5 d; power density, 2366 ± 34 mW m

Microneedle (MN) sensing of biomarkers in interstitial fluid (ISF) can overcome the challenges of self-diagnosis of diseases by a patient, such as blood sampling, handling, and measurement analysis. However, the MN sensing technologies still suffer from poor measurement accuracy due to the small amount of target molecules present in ISF, and require multiple steps of ISF extraction, ISF isolation from MN, and measurement with additional equipment. Here, we present a swellable MN-mounted nanogap sensor that can be inserted into the skin tissue, absorb ISF rapidly, and measure biomarkers in situ by amplifying the measurement signals by redox cycling in nanogap electrodes. We demonstrate that the MN-nanogap sensor measures levodopa (LDA), medication for Parkinson disease, down to 100 nM in an aqueous solution, and 1 μM in both the skin-mimicked gelatin phantom and porcine skin.

In-field screening of pathogenic bacteria is important for preventing food poisoning. Here, a portable pipette-adapted biosensor using magnetic grid separation and nanocatalyst signal amplification was elaboratively developed for rapid detection of Salmonella typhimurium. A common pipette was innovatively adapted with multiple functions to complete the whole bacterial detection procedure, including mixing, separation, catalysis, washing, detection, analysis and display. The target bacteria were effectively captured by the immune magnetic nanobeads and labeled with immune gold@platinum nanocatalysts through pipette-blowing mixing to form the nanobeads-bacteria-nanocatalyst complexes, which were separated against the magnetic grid separation tip under the magnetic field. The pressure change resulting from oxygen production due to mimicking catalysis of hydrogen peroxide by these nanocatalysts on the complexes was quantified through measuring the moving duration of the conductive liquid in the pipette for bacteria determination. Under optimal conditions, this biosensor could detect target bacteria in 90 min with low detection limit of 180 CFU/mL. This pipette-adapted biosensor is affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end-users (ASSURED), and has the potential for in-field testing of foodborne pathogens to ensure food safety, especially in resource-constrained areas.

A novel 3D C-based nano Cu

Innovative optoelectronics are expected to play more important role in clinical diagnosis. In this study, on the basis of sensitive gating effect by in situ enzymatic functionalization of semiconductors, a novel organic photoelectrochemical transistor (OPECT) detection of serum alkaline phosphatase (ALP) level was demonstrated. Specifically, the OPECT detection operates upon the ALP-catalyzed hydrolysis of sodium thiophosphate to yield hydrogen sulfide (H

The commercialization of amperometric or voltammetric biosensors that operate at potentials lower than -0.2 V vs SHE has been hindered by the need for anoxic working conditions due to the interference of molecular oxygen, whose electrochemical reduction can potentially mask other redox processes and generate reactive oxygen species (ROS). A deoxygenation step must be thus integrated into the analytical process. To this end, several (bio)chemical oxygen scavenging systems have been proposed, such as the bi-enzyme system, glucose oxidase/catalase. Still, a few issues persist owing to enzyme impurities and the formation of oxygen reactive species. Here in, we propose a new mono-enzymatic oxygen scavenging system composed of a multicopper oxidase as a single biocatalytic oxygen reducer. As a model, we used bilirubin oxidase (BOD), which catalyzes the direct reduction of oxygen to water in the presence of an electron donor substrate, without releasing hydrogen peroxide. Both the direct electron transfer and mediated electrochemical approach using different co-substrates were screened for the ability to promote the enzymatic reduction of oxygen. An optimal combination of BOD with sodium ascorbate proved to be quick (5 min) and effective. It was subsequently employed, as a proof-of-concept, in a voltammetric biosensor based on a multiheme cytochrome c nitrite reductase, which performs the reduction of nitrite to ammonia at potentials below -0.3 V vs SHE. The nitrite biosensor performed well under ambient air, with no need for a second enzyme to account for the build-up of oxygen reactive intermediaries.