Monosaccharide-Mediated Rational Synthesis of a Universal Plasmonic Platform With Broad Spectral Fluorescence Enhancement For High-Sensitivity Cancer Biomarker Analysis

BackgroundEffective and accurate screening of oncological biomarkers in peripheral blood circulation plays an increasingly vital role in diagnosis and prognosis. High-sensitivity assays can effectively aid clinical decision-making and intervene in cancer in a localized status before they metastasize and become unmanageable. Meanwhile, it is equally pivotal to prevent overdiagnosis of non-life-threatening cancer by eliminating unnecessary treatment and repeated blood draws. Unfortunately, current clinical screening methodologies can hardly simultaneously attain sufficient sensitivity and specificity, especially under resource-restrained circumstances. To circumvent such limitations, particularly for cancer biomarkers from early-onset and recurrence, we aim to develop a universal plasmonic platform for clinical applications, which macroscopically amplifies multiplexed fluorescence signals in a broad spectral window readily adapts to current assay setups without sophisticated accessories or expertise at low cost. MethodsThe plasmonic substrate was chemically synthesized in situ at the solid-liquid interface by rationally screening a panel of reducing monosaccharides and tuning the redox reactions at various catalyst densities and precursor concentrations. The redox properties were studied by Benedict’s assay and electrochemistry. We systemically characterized the morphologies and optical properties of the engineered plasmonic Ag structures by scanning electron microscopy (SEM) and spectroscopy. The structure-fluorescence enhancement correlation was explicitly explained by the finite-difference time-domain (FDTD) simulation and a computational model for gap distribution. Next, we established an enhanced fluoroimmunoassay (eFIA) using a model biomarker for prostate cancer (PCa) and validated it in healthy and PCa cohorts. Prognosis was explored in patients subject to surgical and hormonal interventions following recommended PCa guidelines. ResultsThe monosaccharide-mediated redox reaction yielded a broad category of Ag structures, including sparsely dispersed nanoparticles of various sizes, semi-continuous nanoislands, and crackless continuous films. Optimal broad-spectral fluorescence enhancement from green to far-red was observed for the inhomogeneous, irregularly-shaped semi-continuous Ag nanoisland substrate (AgNIS), synthesized from a well-balanced redox reaction at a stable rate mediated by mannose. In addition, different local electric field intensity distributions in response to various incident excitations were observed at the nanoscale, elucidating the need for irregular and inhomogeneous structures. AgNIS enabled a maximized 54.7-fold macroscopically amplified fluorescence and long-lasting photostability. Point-of-care availability was fulfilled using a customized smartphone prototype with well-paired optics. The eFIA effectively detected the PCa marker in cell lines, xenograft tumors, and patient sera. The plasmonic platform rendered a diagnostic sensitivity of 86.0% and a specificity of 94.7% and capably staged high-grade PCa that the clinical gold standard test failed to stratify. Patient prognosis of surgical and hormonal interventions was non-invasively monitored following efficient medical interventions. The assay time was significantly curtailed on the plasmonic platform upon microwave irradiation. ConclusionsBy investigating the effects of monosaccharides on the seed-mediated chemical synthesis of plasmonic Ag structures, we deduced that potent multiplexed fluorescence enhancement originated from both an adequate reducing power and a steady reduction rate. Furthermore, the inhomogeneous structure with adequate medium gap distances afforded optimal multiwavelength fluorescence enhancement, thus empowering an effective eFIA for PCa. The clinically validated diagnostic and prognostic features, along with the low sample volume, point-of-care feasibility with a smartphone, and microwave-shortened assay time, warrant its potential clinical translation for widespread cancer biomarker analysis.

An Origami Paper-Based Analytical Device for Rapid and Sensitive Analysis of Acrylamide in Foods

Rapid and sensitive detection of acrylamide in food samples is important for food safety and public health. Here, we describe a disposable origami paper-based analytical device (denoted doPAD) for colorimetric detection of acrylamide. This device uniquely exploits 3D origami folding paper for spatial control of the target recognition and signal readout, thus resulting in a positive correlation between the signals and the analytes. Under optimal conditions, the device achieved the quantitative analysis of acrylamide with a limit of detection of 1.13 μg/L within 120 min (including a derivatization time of 90 min and an assay time of 21 min). Furthermore, our method allowed the rapid and sensitive detection of acrylamide in complex food matrices. We envision that the platform described will find useful applications in the fields of food safety and environmental health.

Biosensors as Nano-Analytical Tools for COVID-19 Detection

Selective, sensitive and affordable techniques to detect disease and underlying health issues have been developed recently. Biosensors as nanoanalytical tools have taken a front seat in this context. Nanotechnology-enabled progress in the health sector has aided in disease and pandemic management at a very early stage efficiently. This report reflects the state-of-the-art of nanobiosensor-based virus detection technology in terms of their detection methods, targets, limits of detection, range, sensitivity, assay time, etc. The article effectively summarizes the challenges with traditional technologies and newly emerging biosensors, including the nanotechnology-based detection kit for COVID-19; optically enhanced technology; and electrochemical, smart and wearable enabled nanobiosensors. The less explored but crucial piezoelectric nanobiosensor and the reverse transcription-loop mediated isothermal amplification (RT-LAMP)-based biosensor are also discussed here. The article could be of significance to researchers and doctors dedicated to developing potent, versatile biosensors for the rapid identification of COVID-19. This kind of report is needed for selecting suitable treatments and to avert epidemics.

Study on Factors Affecting the Performance of a CRISPR/Cas-Assisted New Immunoassay: Detection of Salivary Insulin as an Example

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas is now playing a significant role in biosensing applications, especially when the trans-cleavage activity of several Cas effectors is discovered. Taking advantages of both CRISPR/Cas and the enzyme-linked immunosorbent assay (ELISA) in analytical and clinical investigations, CRISPR/Cas-powered ELISA has been successfully designed to detect a spectrum of analytes beyond nucleic acid. Herein, we developed a CRISPR/Cas12a-assisted new immunoassay (CANi) for detection of salivary insulin as an example. Specifically, factors (antibody selection, temperature, and assay time) affecting the CRISPR/Cas-based ELISA system’s performance were investigated. It was observed that the concentration of blocking solution, selection of the capture antibody pairs, and the sequences of triggering ssDNA and guiding RNA affected this immunoassay sensitivity. In contrast, the preincubation of CRISPR/Cas12a working solution and pre-mixture of detection antibody with anti-IgG–ssDNA did not show influence on the performance of CANi for the detection of insulin. Under optimized conditions, the sensitivity for detection of salivary insulin was 10 fg/ml with a linear range from 10 fg/ml to 1 ng/ml.

Voltammetric Immunosensor to Track a Major Peanut Allergen (Ara h 1) in Food Products Employing Quantum Dot Labels

Tracking unreported allergens in commercial foods can avoid acute allergic reactions. A 2-step electrochemical immunosensor was developed for the analysis of the peanut allergen Ara h 1 in a 1-h assay (<15 min hands-on time). Bare screen-printed carbon electrodes (SPCE) were used as transducers and monoclonal capture and detection antibodies were applied in a sandwich-type immunoassay. The short assay time was achieved by previously combining the target analyte and the detection antibody. Core/shell CdSe@ZnS Quantum Dots were used as electroactive label for the detection of the immunological interaction by differential pulse anodic stripping voltammetry. A linear range between 25 and 1000 ng·mL−1 (LOD = 3.5 ng·mL−1), an adequate precision of the method (Vx0 ≈ 6%), and a sensitivity of 23.0 nA·mL·ng−1·cm−2 were achieved. The immunosensor was able to detect Ara h 1 in a spiked allergen-free product down to 0.05% (m/m) of peanut. Commercial organic farming cookies and cereal and protein bars were tested to track and quantify Ara h 1. The results were validated by comparison with an ELISA kit.

Short Assay Design for Micronucleus Detection in Human Lymphocytes

There has been a constant need to develop new and faster cytogenetic assays to measure the instability induced by genotoxic agents in the field of cytogenetic research, an example of which is the micronucleus assay. Micronuclei are fragments or complete chromosomes that remain in the cytoplasm during mitosis. With their high sensitivity and specificity detection, their presence can indicate environmental and occupational genotoxic effects. However, the prolonged periods of cell incubation this assay necessitates are costly and extensive. Hence, it is essential to develop an improved assay that can achieve standardization by being reproducible in practice. The standard protocol for the detection of micronuclei in lymphocytes uses a total assay time of 72 hours. Theoretically, it is possible to reduce the incubation period, and consequently, the total assay time, considering a lymphocyte, completes its mitosis in 24 hours. This study, after careful review of literature, proposes an experimental design to reduce the incubation period and demonstrates its usefulness in practice through the design of a collaborative trial.

Cocaine Detection by a Laser-Induced Immunofluorometric Biosensor

The trafficking of illegal drugs by criminal networks at borders, harbors, or airports is an increasing issue for public health as these routes ensure the main supply of illegal drugs. The prevention of drug smuggling, including the installation of scanners and other analytical devices to detect small traces of drugs within a reasonable time frame, remains a challenge. The presented immunosensor is based on a monolithic affinity column with a large excess of immobilized hapten, which traps fluorescently labeled antibodies as long as the analyte cocaine is absent. In the presence of the drug, some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein, detectable by highly sensitive laser-induced fluorescence with the help of a Peltier-cooled complementary metal-oxide-semiconductor (CMOS) camera. Liquid handling is performed with high-precision syringe pumps and microfluidic chip-based mixing devices and flow cells. The biosensor achieved limits of detection of 7 ppt (23 pM) of cocaine with a response time of 90 s and a total assay time below 3 min. With surface wipe sampling, the biosensor was able to detect 300 pg of cocaine. This immunosensor belongs to the most sensitive and fastest detectors for cocaine and offers near-continuous analyte measurement.

Cocaine Detection by a Laser-induced Immunofluorometric Biosensor

The trafficking of illegal drugs by criminal networks at borders, harbors, or airports is an increasing issue in public health as these routes ensure the main supply of illegal drugs. The prevention of drug smuggling, including the installation of scanners and other analytical devices to detect ultra-small traces of drugs within a reasonable time frame, remains a challenge. The presented immunosensor is based on a monolithic affinity column with a large excess of immobilized hapten, which traps fluorescently labeled antibodies as long as the analyte cocaine is absent. In the presence of the drug, some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein, detectable by highly sensitive laser-induced fluorescence with the help of a Peltier-cooled complementary metal-oxide-semiconductor (CMOS) camera. Liquid handling is performed with high-precision syringe pumps and microfluidic chip-based mixing devices and flow cells. The biosensor achieved limits of detection of 23 pM (7 ppt) of cocaine with a response time of 90 seconds and a total assay time below 3 minutes. With surface wipe sampling, the biosensor was able to detect 300 pg of cocaine. This immunosensor belongs to the most sensitive and fastest detectors for cocaine and offers near-continuous analyte measurement.

Tropomyosin Analysis in Foods Using an Electrochemical Immunosensing Approach

A screen-printed carbon electrode was used as the transducer for the development of an electrochemical immunosensor for the determination of tropomyosin (a major shrimp allergen) in food samples. Monoclonal and polyclonal antibodies were used in a sandwich-type immunoassay. The analytical signal was electrochemically obtained using an alkaline phosphatase-labelled secondary antibody and a 3-indoxyl phosphate/silver nitrate substrate. The total assay time was 2 h 50 min and allowed the quantification of tropomyosin between 2.5 and 20 ng mL−1, with a limit of detection of 1.7 ng mL−1 The immunosensor was successfully applied to the analysis of commercial food products.

Ti3C2 MXene-Based Nanobiosensors for Detection of Cancer Biomarkers

This chapter provides information about basic properties of MXenes (2D nanomaterials) that are attractive for a design of various types of nanobiosensors. The second part of the chapter discusses MXene synthesis and various protocols for modification of MXene making it a suitable matrix for immobilization of bioreceptors such as antibodies, DNA aptamers or DNA molecules. The final part of the chapter summarizes examples of MXene-based nanobiosensors developed using optical, electrochemical and nanomechanical transducing schemes. Operational characteristics of such devices such as sensitivity, limit of detection, assay time, assay reproducibility and potential for multiplexing are provided. In particular MXene-based nanobiosensors for detection of a number of cancer biomarkers are shown here.