Sub-picomolar Detection Limit Research Articles

CRISPR-Cas12a-regulated DNA adsorption and metallization on MXenes as enhanced enzyme mimics for sensitive colorimetric detection of hepatitis B virus DNA

Hepatitis B virus (HBV) infection is closely associated with the high risk of evolving into human hepatitis diseases including chronic hepatitis, liver fibrosis and cirrhosis, as well as hepatoma. Although various methods have been developed for HBV DNA detection, most of them either rely on expensive instruments or laborious procedures involving professional personnel. In this study, we for the first time established the CRISPR-Cas12a based colorimetric biosensor for target HBV detection by utilizing probe DNA regulation of the catalytic behaviors of Mxene-probe DNA-Ag/Pt nanohybrids. In the presence of HBV target, the Cas12a trans-cleavage activity could be efficiently activated to degrade the DNA probes, which led to the inhibition of DNA metallization and enzyme activity enhancer DNA adsorbed on Mxene, resulting in significantly reduced catalytic activity. The Mxene-probe DNA-Ag/Pt nanohybrids exhibited excellent sensitivity and specificity with subpicomolar detection limits, as well as good accuracy and stability for the determination of target HBV DNA in human serum samples. Moreover, this colorimetric sensing strategy could be integrated with the smartphone platform to allow the visible sensitive detection of target DNA. Taken together, the proposed colorimetric method provides a novel approach for HBV DNA diagnosis, especially suitable for the high endemic, developing countries with limited instrumental and medical supports.

Highly sensitive and quantitative biodetection with lipid-polymer hybrid nanoparticles having organic room-temperature phosphorescence

A versatile organic room-temperature phosphorescence (RTP)-based “turn on” biosensor platform has been devised with high sensitivity by combining oxygen-sensitive lipid-polymer hybrid RTP nanoparticles with a signal-amplifying enzymatic oxygen scavenging reaction in aqueous solutions. When integrated with a sandwich-DNA hybridization assay on 96-well plates, our phosphorimetric sensor demonstrates sequence-specific detection of a cell-free cancer biomarker, a TP53 gene fragment, with a sub-picomolar (0.5 p.m.) detection limit. This assay is compatible with detecting cell-free nucleic acids in human urine samples. Simply by re-programming the detection probe, our unique methodology can be adapted to a broad range of biosensor applications for biomarkers of great clinical importance but difficult to detect due to their low abundance in vivo.

Immunoglobulin G-Based Steric Hindrance Assay for Protein Detection.

With the imminent needs of rapid, accurate, simple point-of-care systems for global healthcare industry, electrochemical biosensors have been widely developed owing to their cost-effectiveness and simple instrumentation. However, typical electrochemical biosensors for direct analysis of proteins in the human biological sample still suffer from complex biosensor fabrication, lack of general method, limited sensitivity, and matrix-caused biofouling effect. To resolve these challenges, we developed a general electrochemical sensing strategy based on a designed steric hindrance effect on an antibody surface layer. This strategy utilizes the interaction pattern of protein-G and immunoglobulin G (Fc and Fab regions), providing a steric hindrance effect during the target capturing process. The provided steric hindrance effect minimizes the matrix effect-caused fouling surface and altered the path of electron transfer, delivering a low-fouling and high-sensitivity detection of protein in complex matrices. Also, an enzyme-based horseradish peroxidase/hydroquinone/H2O2 transduction system can also be applied to the system, demonstrating the versatility of this sensing strategy for general electrochemical sensing applications. We demonstrated this platform through the detection of Tau protein and programming death ligand 1 with a subpico molar detection limit within 10 min, satisfying the clinical point-of-care requirements for rapid turnaround time and ultrasensitivity.

Single-Molecule Topochemical Analyses for Large-Scale Multiplexing Tasks.

Multitasking is the pivotal feature in next-generation chemo- or bioanalyses. However, simultaneous analyses rarely exceed over three different tasks, which is ascribed to the limited space to accommodate analyzing units and the compromised signal-to-noise (S/N) level as the number of tasks increases. Here, by leveraging superior S/N of single-molecule techniques, we analyzed five microRNA biomarkers by spatially encoding miRNA recognition units with nanometers resolution in a DNA template, while decoding the analyte binding temporally in seconds. The hairpin stem is interspersed by internal loops to encode recognition units for miRNA. By mechanical unfolding of the hairpin, individual internal loops are sequentially interrogated for the binding of each miRNA. Using this so-called topochemical spatiotemporal analysis, we were able to achieve subpicomolar detection limits of miRNAs. We anticipate that this new single-molecule topochemical analysis can massively analyze single-molecule targets.

Challenges and Solutions in Developing Ultrasensitive Biosensors.

This Perspective focuses on the latest strategies and challenges for the development of bioanalytical sensors with sub-picomolar detection limits. Achieving sub-picomolar detection limits has three major challenges: (1) assay sensitivity, (2) response time, and (3) selectivity (including limiting background signals). Each of these challenges is discussed, along with how nanomaterials provide the solutions. One strategy to gain greater sensitivity involves confining the sensing volume to the nanoscale, as used in nanopore- or nanoparticle-based sensors, because nanoparticles are ubiquitous in amplification. Methods to improve response time typically focus on obtaining an intimate mixture between the sensor and the sample either by extending the length scale of nanoscale sensors using nanostructuring or by dispersing magnetic nanoparticles through the sample to capture the analyte. Loading nanoparticles with many biorecognition species is one solution to help address the challenge of selectivity. Many examples in this Perspective explore the detection of prostate-specific antigenwhich enables a comparison between strategies. Finally, exciting future opportunities in developing single-molecule sensors and the requirements to go even lower in concentration are explored.

Direct, Label-Free, and Rapid Transistor-Based Immunodetection in Whole Serum.

Transistor-based biosensors fulfill many requirements posed upon transducers for future point-of-care diagnostic devices such as scalable fabrication and label-free and real-time quantification of chemical and biological species with high sensitivity. However, the short Debye screening length in physiological samples (<1 nm) has been a major drawback so far, preventing direct measurements in serum. In this work, we demonstrate how tailoring the sensing surface with short specific biological receptors and a polymer polyethylene glycol (PEG) can strongly enhance the sensor response. In addition, the sensor performance can be dramatically improved if the measurements are performed at elevated temperatures (37 °C instead of 21 °C). With this novel approach, highly sensitive and selective detection of a representative immunosensing parameter-human thyroid-stimulating hormone-is shown over a wide measuring range with subpicomolar detection limits in whole serum. To the best of our knowledge, this is the first demonstration of direct immunodetection in whole serum using transistor-based biosensors, without the need for sample pretreatment, labeling, or washing steps. The presented sensor is low-cost, can be easily integrated into portable diagnostics devices, and offers a competitive performance compared to state-of-the-art central laboratory analyzers.

Highly sensitive, selective and label-free protein detection in physiological solutions using carbon nanotube transistors with nanobody receptors

Nanomaterial-based field-effect transistors (FETs) have been proposed for real-time, label-free detection of various biological species. However, two major challenges have limited their use in physiological samples: screening of the analyte charge by electrolyte ions (Debye screening) and non-specific adsorption. Here, these challenges are overcome by combining highly stable FETs based on single-walled semiconducting carbon nanotube (SWCNTs) networks with a novel surface functionalization comprising: 1) short nanobody (VHH) receptors, and 2) a polyethylene glycol (PEG) layer. Nanobodies are stable, easy-to-produce biological receptors that are very small (∼2–4nm), thus enabling analyte binding closer to the sensor surface. Despite their unique properties, nanobodies have not been used yet as receptors in FET based biosensors. The addition of PEG strongly enhances the signal in high ionic strength environment. Using green fluorescent protein (GFP) as a model antigen, high selectivity and sub-picomolar detection limit with a dynamic range exceeding 5 orders of magnitude is demonstrated in physiological solutions. In addition, long-term stability measurements reveal a low drift of SWCNTs of 0.05mV/h. The presented immunoassay is fast, label-free, does not require any sample pretreatment or washing steps.

A Guide to Quantitative Biomarker Assay Development using Whispering Gallery Mode Biosensors.

Whispering gallery mode (WGM) sensors are a class of powerful analytical techniques defined by the measurement of changes in the local refractive index at or near the sensor surface. When functionalized with target-specific capture agents, analyte binding can be measured with very low limits of detection. There are many geometric manifestations of WGM sensors, with chip-integrated silicon photonic devices first commercialized because of the robust, wafer-scale device fabrication, facile optical interrogation, and amenability to the creation of multiplexed sensor arrays. Using these arrays, a number of biomolecular targets have been detected in both label-free and label-enhanced assay formats. For example, sub-picomolar detection limits for multiple cytokines were achieved using an enzymatically enhanced sandwich immunoassay that showed high analyte specificity suitable for detection in complex, clinical matrices. This protocol describes a generalizable approach for the development of quantitative, multiplexed immunoassays using silicon photonic microrings as an example WGM platform. © 2017 by John Wiley & Sons, Inc.

Peptide-Templated Gold Nanocluster Beacon as a Sensitive, Label-Free Sensor for Protein Post-translational Modification Enzymes

Protein post-translational modifications (PTMs), which are chemical modifications and most often regulated by enzymes, play key roles in functional proteomics. Detection of PTM enzymes, thus, is critical in the study of cell functioning and development of diagnostic and therapeutic tools. Herein, we develop a simple peptide-templated method to direct rapid synthesis of highly fluorescent gold nanoclusters (AuNCs) and interrogate the effect of enzymatic modifications on their luminescence. A new finding is that enzymes are able to exert chemical modifications on the peptide-templated AuNCs and quench their fluorescence, which furnishes the development of a real-time and label-free sensing strategy for PTM enzymes. Two PTM enzymes, histone deacetylase 1 and protein kinase A, have been employed to demonstrate the feasibility of this enzyme-responsive fluorescent nanocluster beacon. The results reveal that the AuNCs' fluorescence can be dynamically decreased with increasing concentration of the enzymes, and subpicomolar detection limits are readily achieved for both enzymes. The developed strategy can thus offer a useful, label-free biosensor platform for the detection of protein-modifying enzymes and their inhibitors in biomedical applications.

Second harmonic generation correlation spectroscopy for single molecule experiments

We demonstrate a single molecule detection approach to further extend the detection limit of correlation spectroscopic techniques through the Second Harmonic Generation Correlation Spectroscopy (SHGCS). SHG signals with high signal-to-noise ratio (SNR) were obtained from Barium titanium oxide, BaTiO(3) (BTO) nanocrystals (NCs) upon excitation by a femto-second laser fitted to the scanning confocal bench. The fluctuation of SHG signals from BTO NCs in transparent and turbid media was examined and their diffusion time and particle concentration were evaluated by autocorrelation. Proof-of-concept measurements indicate that water-dispersed BTO NCs at different concentrations yield an average diffusion time of 6.43 ± 0.68 ms and the detection limit of SHGCS was found to be at 814 ± 41 fM, approximately 100 folds below the detection limit of fluorescence correlation spectroscopy (FCS). The dynamics of BTO NCs was demonstrated in serum with high SNR and selectivity to show its potential applicability in biomedicine. High SNR and the sub-picomolar detection limit positions SHGCS as an excellent technique for ultralow single particle or single molecule experimentation in a complex medium.

An exonuclease III and graphene oxide-aided assay for DNA detection

We have developed a novel DNA assay based on exonuclease III (ExoIII)-induced target recycling and the fluorescence quenching ability of graphene oxide (GO). This assay consists of a linear DNA probe labeled with a fluorophore in the middle. Introduction of target sequence induces the exonuclease III catalyzed probe digestion and generation of single nucleotides. After each cycle of digestion, the target is recycled to realize the amplification. Finally, graphene oxide is added to quench the remaining probes and the signal from the resulting fluorophore labeled single nucleotides is detected. With this approach, a sub-picomolar detection limit can be achieved within 40min at 37°C. The method was successfully applied to multicolor DNA detection and the analysis of telomerase activity in extracts from cancer cells.

Patterning of Quantum Dot Bioconjugates via Particle Lithography

We present a simple technique to fabricate hexagonally ordered quantum dot bioconjugate (QDBC) dot arrays on glass coverslips. We used particle lithography to create periodic holes in a layer of methoxy-poly(ethylene glycol)-silane and then adsorbed QDBCs into the holes. To demonstrate the versatility of this technique, we made separate periodic arrays of quantum dots (QDs) conjugated to three different biologically important molecules: biotin, streptavidin, and anti-mouse IgG. The diameters of the regions where the QDBCs adsorbed were 500-600 nm and independent of the QDBC patterned. The site density of the QDBCs in the patterned holes could be varied by simply adjusting the coating concentration of the QDBC solution. We demonstrate the applicability of these substrates by designing a QDBC-based binding assay with a working concentration range of several orders of magnitude and a sub-picomolar detection limit.

Quantum Dot Biosensors for Ultrasensitive Multiplexed Diagnostics

Colorful bioassays: Time- and color-resolved detection of Förster resonance energy transfer (FRET) from luminescent terbium complexes to different semiconductor quantum dots results in a fivefold multiplexed bioassay with sub-picomolar detection limits for all five bioanalytes (see picture). The detection of up to five biomarkers occurs with a sensitivity that is 40–-240-fold higher than one of the best-established single-analyte reference assays.

Single-step electronic detection of femtomolar DNA by target-induced strand displacement in an electrode-bound duplex.

We report a signal-on, electronic DNA (E-DNA) sensor that is label-free and achieves a subpicomolar detection limit. The sensor, which is based on a target-induced strand displacement mechanism, is composed of a "capture probe" attached by its 5' terminus to a gold electrode and a 5' methylene blue-modified "signaling probe" that is complementary at both its 3' and 5' termini to the capture probe. In the absence of target, hybridization between the capture and signaling probes minimizes contact between the methylene blue and electrode surface, limiting the observed redox current. Target hybridization displaces the 5' end of the signaling probe, generating a short, flexible single-stranded DNA element and producing up to a 7-fold increase in redox current. The observed signal gain is sufficient to achieve a demonstrated (not extrapolated) detection limit of 400 fM, which is among the best reported for single-step electronic DNA detection. Moreover, because sensor fabrication is straightforward, the approach appears to provide a ready alternative to the more cumbersome femtomolar electrochemical assays described to date.

Fluorescence of Aromatic Amines and Their Fluorescamine Derivatives for Detection of Explosive Vapors

Nitroaromatics (such as dinitrotoluene, trinitrotoluene, and nitrobenzene) found in explosive vapors from buried landmines can be reduced to aminoaromatics by a novel process involving Pd metal nanocatalysts prepared in supercritical fluid carbon dioxide and supported on multi-walled carbon nanotubes. These aminoaromatics are fluorescent and, if desired, the fluorescence yield can be increased and the fluorescence maxima shifted further toward the red by reaction with appropriate derivatizing agents such as fluorescamine. Corrected spectra for these chemicals and their derivatives are included. Subpicomolar detection limits have already been achieved using a laboratory spectrofluorometer with a 150 W Xe arc lamp. Using lasers as excitation sources, this approach has the potential for developing a field sensor competitive with other methods currently used for detecting explosive vapors from land mines.

Fundamentals and practice for ultrasensitive laser‐induced fluorescence detection in microanalytical systems

Laser-induced fluorescence is an extremely sensitive method for detection in chemical separations. In addition, it is well-suited to detection in small volumes, and as such is widely used for capillary electrophoresis and microchip-based separations. This review explores the detailed instrumental conditions required for sub-zeptomole, sub-picomolar detection limits. The key to achieving the best sensitivity is to use an excitation and emission volume that is matched to the separation system and that, simultaneously, will keep scattering and luminescence background to a minimum. We discuss how this is accomplished with confocal detection, 90 degrees on-capillary detection, and sheath-flow detection. It is shown that each of these methods have their advantages and disadvantages, but that all can be used to produce extremely sensitive detectors for capillary- or microchip-based separations. Analysis of these capabilities allows prediction of the optimal means of achieving ultrasensitive detection on microchips.

Determination of picomolar levels of flavins in natural waters by solid-phase ion-pair extraction and liquid chromatography

A method is described for the rapid determination of flavins in sea water, based on solid-phase extraction followed by ion-pair high-performance liquid chromatography (HPLC) with fluorescence detection. Riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) and their photochemical breakdown products, lumiflavin, formylmethylflavin, and lumichrome can be determined with subpicomolar detection limits. The method was used at sea in the analysis of coastal and open ocean waters. In both environments, riboflavin, lumiflavin and lumichrome were routinely observed at concentrations in the picomolar range; lumiflavin and lumichrome were generally confined to the photic zone while riboflavin was present throughout the water column. Formylmethylflavin, FMN, and FAD were only occasionally observed; when present, these flavins were observed at consistently higher concentrations than riboflavin, lumiflavin and lumichrome.