Improved Specificity For Detection Research Articles

Reliability of lab-on-a-chip technologies for wearable electronics: a perspective

Over the past few decades, Lab-on-a-chip (LoC) devices have made health diagnostics easier and cheaper by enabling rapid, hassle-free, and inexpensive measurements of different biochemical markers, properties of epidermally retrievable biofluids (e.g., sweat and interstitial fluids), etc. The miniaturization of these sensing technologies along with the integration of flexible frameworks (microfluidic assemblies and electronics) and user-friendly software have paved the way for autonomous and continuous tracking of human health parameters. However, with emerging new technologies, there are new and complex challenges, especially in the case of these miniature LoC devices. Although microfluidics-specific challenges such as analyte contamination, flow rate variability, improving detection specificity, etc. are being worked upon, the bigger question is: how reliable are these wearable devices in the long run? This perspective piece initially talks about the state-of-the-art LoC technologies that have been incorporated into wearable devices. Next, the perspective identifies some of the reliability studies conducted (as well as overlooked) on the integrated wearable electronics used. Finally, the paper discusses the challenges and future directions of research in the field of reliability of LoC-based wearable electronics.

Advances and Trends in miRNA Analysis Using DNAzyme-Based Biosensors.

miRNAs are endogenous small, non-coding RNA molecules that function in post-transcriptional regulation of gene expression. Because miRNA plays a pivotal role in maintaining the intracellular environment, and abnormal expression has been found in many cancer diseases, detection of miRNA as a biomarker is important for early diagnosis of disease and study of miRNA function. However, because miRNA is present in extremely low concentrations in cells and many types of miRNAs with similar sequences are mixed, traditional gene detection methods are not suitable for miRNA detection. Therefore, in order to overcome this limitation, a signal amplification process is essential for high sensitivity. In particular, enzyme-free signal amplification systems such as DNAzyme systems have been developed for miRNA analysis with high specificity. DNAzymes have the advantage of being more stable in the physiological environment than enzymes, easy to chemically synthesize, and biocompatible. In this review, we summarize and introduce the methods using DNAzyme-based biosensors, especially with regard to various signal amplification methods for high sensitivity and strategies for improving detection specificity. We also discuss the current challenges and trends of these DNAzyme-based biosensors.

Deploying wearable sensors for pandemic mitigation: A counterfactual modelling study of Canada's second COVID-19 wave.

Wearable sensors can continuously and passively detect potential respiratory infections before or absent symptoms. However, the population-level impact of deploying these devices during pandemics is unclear. We built a compartmental model of Canada's second COVID-19 wave and simulated wearable sensor deployment scenarios, systematically varying detection algorithm accuracy, uptake, and adherence. With current detection algorithms and 4% uptake, we observed a 16% reduction in the second wave burden of infection; however, 22% of this reduction was attributed to incorrectly quarantining uninfected device users. Improving detection specificity and offering confirmatory rapid tests each minimized unnecessary quarantines and lab-based tests. With a sufficiently low false positive rate, increasing uptake and adherence became effective strategies for scaling averted infections. We concluded that wearable sensors capable of detecting presymptomatic or asymptomatic infections have potential to help reduce the burden of infection during a pandemic; in the case of COVID-19, technology improvements or supporting measures are required to keep social and resource costs sustainable.

Single-Molecule Counting of FTO in Human Breast Tissues Based on a Rolling Circle Transcription Amplification-Driven Clustered Regularly Interspaced Short Palindromic Repeat─Cas12a.

N6-methyladenosine modification as an mRNA modification in mammalian cells is dynamically reversible, regulated by RNA demethylase [e.g., fat mass and obesity-associated protein (FTO)]. The abnormal expression of FTO is closely related to numerous diseases (e.g., various cancers and obesity). Herein, we demonstrate the single-molecule counting of FTO in human cancer cells and breast tissues based on a T7 RNA polymerase-mediated rolling circle transcription (RCT) amplification-driven clustered regularly interspaced short palindromic repeat (CRISPR)─Cas12a. When FTO is present, it demethylates the DNA substrate, initiating the DpnII-mediated cleavage reaction. After magnetic separation, the cleaved DNA fragments trigger the T7 RNA polymerase-mediated RCT amplification, activating CRISPR-/Cas12a-mediated cleavage of signal probes and releasing abundant FAM molecules that are simply counted via single-molecule detection. In this assay, only target FTO can generate CRISPR RNAs, efficiently improving detection specificity. Moreover, the integration of single-molecule detection with magnetic separation achieves zero background and effectively enhances detection sensitivity. This method can specifically and sensitively monitor FTO activity with a limit of detection of 1.20 × 10-13 M, and it may measure FTO at the single-cell level. Furthermore, it may accurately discriminate the FTO expression level in breast tissues between healthy persons and breast cancer patients and screen the FTO inhibitors as well, with great potential in clinical diagnosis and drug discovery.

Quantitative and ultrasensitive in situ immunoassay technology for SARS-CoV-2 detection in saliva.

The coronavirus disease 2019 (COVID-19) pandemic has become an immense global health crisis. However, the lack of efficient and sensitive on-site testing methods limits early detection for timely isolation and intervention. Here, we present a quantitative and ultrasensitive in situ immunoassay technology for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in saliva (QUIT SARS-CoV-2). Our nanoporous membrane resonator generates a rapid oscillating flow to purify and concentrate fully intact SARS-CoV-2 virus in saliva by 40-fold for in situ detection of viral antigens based on chemiluminescent immunoassay within 20 min. This method can not only achieve a detection sensitivity below 100 copies/ml of virus, comparable to the bench-top PCR equipment; it can also improve detection specificity via direct monitoring of viral loads. The integrated portable QUIT SARS-CoV-2 system, which enables rapid and accurate on-site viral screening with a high-throughput sample pooling strategy, can be performed in primary care settings and substantially improve the detection and prevention of COVID-19.

Construction of Dual-Target Recognition-Based Specific MicroRNA Detection Method for Acute Pancreatitis Analysis.

MicroRNAs (miRNAs) play crucial roles in regulating various biological processes and are considered promising biomarkers for clinical diagnosis and therapy of acute pancreatitis. Herein, we present a duplex-specific nuclease (DSN enzyme) and DNAzyme-assisted fluorescent miRNA detections assay that can provide improved detection specificity due to a design of dual-target recognition and a comparable sensitivity. The dual-target recognitions are composed of (i) miRNA unfold hairpin structure toehold to form DNA-RNA duplex, among which the DNA section will be digested by DSN enzyme, releasing miRNA to participant in a next recycle. (ii) After DNAzyme-based nicking site formation in loop section of molecular beacon (MB), miRNA can bind with the loop section of MB and gradually unfold MB probe, generating fluorescence signals. With this general principle, distinct discrimination capability towards even one base pair mismatch of homogenous miRNA is obtained, showing a promising prospect in clinical diagnosis and therapy of acute pancreatitis.

Visualization of copper nanoclusters for SARS-CoV-2 Delta variant detection based on rational primers design.

Here, based on the design of rational primers and copper nanoclusters (CuNCs), we present a method for the accurate detection of the SARS-CoV-2 Delta variant, which is capable of distinguishing the Delta variant with its single nucleotide polymorphism from the ‘wild type’ coronavirus (NC_045512.2), and realizing visualization signal out. Specifically, we show that dual priming oligonucleotide (DPO) primers and AT primers can be used to distinguish between wild types and mutations of this virus by polymerase chain reaction (PCR) analysis and that visualization can be achieved via the red fluorescence of CuNCs in ultraviolet radiation. Among the results, it was found that the R-1-down (DPO)-6I and F-1-30 AT, with the single nucleotide deletion site designed at the 3′ end of the downstream primer, showed the best specificity towards the Delta variant. Moreover, the use of AT primers increased the AT contents of the PCR products, thus meeting the template requirements generated by the CuNCs. It was also found that the AT primers could assist with improving detection specificity. Finally, we demonstrate that the visualization of the CuNCs-based detection assay exhibited a linear relationship in 0.5 pg μL−1–50 ng μL−1, with a limit of quantitation (LOQ) of 0.5 pg μL−1.

A novel near-infrared fluorescence probe for detecting and imaging Hg2+ in living cells.

Fluorescence imaging, as one of the important means of biological lesion analysis, is widely used in medical analysis. To improve detection specificity, near-infrared emission fluorescent probes have been developed. Sensitive and selective near-infrared (NIR) fluorescent probes for Hg2+ , which is a heavy metal ion harmful to human health, are urgently needed to investigate the physiological toxicity of Hg2+ . The NIR fluorophore based on the traditional structure of rhodamine was prepared by introducing anthocyanin functional groups, and a rhodamine spiro ring structure was constructed to recognize Hg2+ (CCS-Hg). The probe CCS-Hg demonstrated good selectivity and high detection sensitivity for Hg2+ and the most likely mechanism was verified through theoretical calculations. We applied the probe CCS-Hg in the examination of Hg2+ distribution in living cells by NIR fluorescence imaging. This work provides a promising molecular tool for studying the toxicological effects of mercury ions in cell.

Enrichment-Stowage-Cycle Strategy for Ultrasensitive Electrochemiluminescent Detection of HIV-DNA with Wide Dynamic Range.

Sensitive detection of human immunodeficiency virus DNA (HIV-DNA) is essential for timely diagnosis and cure of the illness. Herein, a novel "enrichment-stowage-cycle" strategy was proposed to fabricate a multiple amplified electrochemiluminecence (ECL) biosensor for HIV-DNA detection. On the basis of the enrichment role of magnetic nanobeads, assembly role of copolymer nanospheres and strand displacement amplification (SDA), the processes were named as "enrichment", "stowage", and "cycle", respectively. The method employed electrochemiluminescent nanospheres (ENs) as signal labels by assembling three layers of CdSe/ZnS quantum dots (QDs) onto the surface of copolymer nanospheres. Compared to QDs, the same concentration of ENs can the enhance the ECL intensity by about 11.3-fold. SDA could further amplify the signals by about 3.77-fold, possessing high sensitivity for low-abundant biomarkers detection. The integration of magnetic separation improved detection specificity and stability, making the method possible for practical application. On the basis of magnetic separation, ENs and SDA, the ECL biosensor realized ultrasensitive detection of 39.81 fM HIV-DNA, which was more sensitive than other HIV-DNA analytical methods, with a wide dynamic range of 0.05 pM to 50 nM. The successful detection of HIV-DNA in complex samples with good sensitivity and accuracy indicated its potential utilization in early judgment of diseases and fabrication of signal amplification platforms.

MicroRNA Detection Specificity: Recent Advances and Future Perspective.

MicroRNAs (miRNAs) are a series of promising molecules that could regulate gene expression, while its expression level and type are strictly related to the early diagnosis, targeted therapy, and prognosis of diseases. Improved detection accuracy of miRNAs would lead to unprecedented progress in early treatment. Numerous methods to improve sensitivity have been proposed and confirmed valuable in miRNAs detection recently. However, the research which especially targets at improving detection specificity remains rare. This Feature gives a retrospective summary of the most common detection methods including Northern blot, reverse transcription quantitative polymerase chain reaction, next-generation sequencing, and microarray. Common methods play an important role in many aspects because of maturity and stability. However, the problem of specificity still exists. Then, this Feature summarizes breakthroughs in traditional techniques which are closely related to progressive methods to improve specificity. In addition, considerable studies focusing on improving accuracy are described in detail. Most of them involve the reasonable combination of common methods, and the strategies that contribute to improving accuracy are classified. This Feature aims to illustrate the importance of detection accuracy and explore how to improve specificity on detecting miRNAs, especially homologous families.

Targeted Molecular Imaging of Pancreatic Cancer with a Miniature Endoscope.

It is highly desirable to develop novel approaches to improve patient survival rate of pancreatic cancer through early detection. Here, we present such an approach based on photoacoustic and fluorescence molecular imaging of pancreatic tumor using a miniature multimodal endoscope in combination with targeted multifunctional iron oxide nanoparticles (IONPs). A novel fan-shaped scanning mechanism was developed to minimize the invasiveness for endoscopic imaging of pancreatic tumors. The results show that the enhancements in photoacoustic and fluorescence signals using amino-terminal fragment (ATF) targeted IONPs were ~four to six times higher compared to that using non-targeted IONPs. Our study indicates the potential of the combination of the multimodal photoacoustic-fluorescence endoscopy and targeted multifunctional nanoparticles as an efficient tool to provide improved specificity and sensitivity for pancreatic cancer detection.

Multiplex and accurate quantification of acute kidney injury biomarker candidates in urine using Protein Standard Absolute Quantification (PSAQ) and targeted proteomics

There is a need for multiplex, specific and quantitative methods to speed-up the development of acute kidney injury biomarkers and allow a more specific diagnosis. Targeted proteomic analysis combined with stable isotope dilution has recently emerged as a powerful option for the parallelized evaluation of candidate biomarkers. This article presents the development of a targeted proteomic assay to quantify 4 acute kidney injury biomarker candidates in urine samples. The proteins included in the assessed panel consisted of myo-inositol oxygenase (MIOX), phosphoenolpyruvate carboxykinase 1 (PCK1), neutrophil gelatinase-associated lipocalin (NGAL) and liver fatty acid-binding protein (L-FABP). The proteomic assay combined an antibody-free sample preparation and a liquid chromatography-selected reaction monitoring (LC-SRM) analysis pipeline. For accurate quantification of the selected candidates, we used PSAQ (Protein Standard Absolute Quantification) standards which are isotopically labeled versions of the target proteins. When added directly to the biological samples, these standards improve detection specificity and quantification accuracy. The multiplexed assay developed for the 4 biomarker candidates showed excellent analytical performance, in line with the recommendations of health authorities. Tests on urine from two small patient cohorts and a group of healthy donors confirmed the relevance of NGAL and L-FABP as biomarkers for AKI diagnosis. The assay is readily adaptable to other biomarker candidates and should be very useful for the simultaneous and accurate quantification of multiple biomarkers.

Analysis of Urinary Prostate-Specific Antigen Glycoforms in Samples of Prostate Cancer and Benign Prostate Hyperplasia.

Glycans of prostate-specific antigen (PSA) in prostate cancer were found to be different from that in benign disease. It is difficult to analyze heterogeneous PSA glycoforms in each individual specimen because of low protein abundance and the limitation of detection sensitivity. We developed a method for prostate cancer diagnosis based on PSA glycoforms. Specific glycoforms were screened in each clinical sample based on liquid chromatography-tandem mass spectrometry with ion accumulation. To look for potential biomarkers, normalized abundance of each glycoform in benign prostate hyperplasia (BPH) and in prostate cancer was evaluated. The PSA glycoform, Hex5HexNAc4NeuAc1dHex1, and monosialylated, sialylated, and unfucosylated glycoforms differed significantly between the prostate cancer and BPH samples. The detection sensitivity (87.5%) and specificity (60%) for prostate cancer identification are higher than those of the serum PSA marker. As low as 100 amol PSA could be detected with the ion accumulation method which has not been reported before. The improved detection specificity can help reduce unnecessary examinations.

Differential contrast of gold nanorods in dual-band OCT using spectral multiplexing

In optical coherence tomography (OCT), differential contrast can be generated by resonant nanoparticles using spectral multiplexing. Differential contrast can be of interest for medical applications for improving detection specificity of structures with low endogenous contrast. Differential contrast has been shown using OCT systems with one bandwidth; how- ever, this requires post-processing that is time consum- ing and reduces image resolution. In this study, we used a dual-band OCT prototype system with two far separated bandwidths in the clinically relevant optical window, and in search for the optimal differential contrast-generating particles for this prototype system, three different gold nanorods (AuNR) samples were investigated. The samples with different particle volume, aspect ratio, and absorption-maximum were imaged in a highly scattering phantom and on chicken muscle. In vitro, differential contrast was observed for the nanorods large (NRL) sample having the absorption- maximum within one bandwidth of the OCT and an average length of 75 nm. For the smaller AuNR (48 nm length) with comparable absorption-maximum, the obtained signal intensities were too low for being visible, although differences in signal intensities be- tween both bandwidths could be measured. NRL optimal concentration for differential contrast using this prototype system is between 100 and 500 lg Au/mL (0.51-2.54 mM). These results demonstrate the potential of real-time imaging of differential contrast in dual-band OCT and motivate in vivo application of plasmon resonant AuNR in order to improve the detection sensitivity for structures that are difficult to identify by OCT such as small blood vessels.

Improving detection specificity of iron oxide nanoparticles (IONPs) using the SWIFT sequence with long T2 suppression

In order to improve the detection specificity of iron oxide nanoparticles (IONPs) delivered to tumors, we embedded saturation pulses into the sweep imaging using Fourier transformation (SWIFT) sequence to suppress long T2 tissues and fat. Simulation of the Bloch equation was first conducted to study behavior of the saturation pulses of various lengths under different T2 and off-resonance conditions. MR experiments were then conducted using in vivo mouse xenografts and a phantom consisting of IONPs, vegetable oil, and explanted tumor specimen, without and with long T2 suppression under a 7T magnetic field. For the in vivo study, arginine-glycine-aspartate (RGD) coated 10nm IONPs (RGD-IONPs) were delivered to tumors implanted in nude mice through both intra-tumor and intravenous injections. Histological studies confirmed that RGD-IONPs efficiently homed to tumors through RGD-integrin interaction. Compared to conventional SWIFT, the proposed method resulted in sufficient suppression on long T2 species but less influence on short T2 species. For both the in vivo and ex vivo studies, significantly improved contrast-to-noise ratio (CNR) was achieved between the IONPs and the long T2 species.

Gold nanorods-bombesin conjugate as a potential targeted imaging agent for detection of breast cancer

Photoacoustic imaging (PAI) is a hybrid biomedical imaging modality that offers both strong optical absorption contrast and high ultrasonic resolution. PAI is capable of in vivo molecular imaging, thus facilitating further molecular and cellular characterization of cancer. In this study, Gold nanorods (GNRs) were synthesized and coated with polyethyleneglycol (PEG). Then, the PEG-GNRs were conjugated with bombesin (BBN), a cancer seeking peptide, for production of a potential photoacoustic targeting imaging agent for detection of breast cancer. The optical property, biocompatibility, stability and in vitro/in vivo binding affinities of GNR-PEG-BBN for breast cancer cells were investigated. UV–vis spectroscopy confirmed the conjugation of bombesin with PEG-coated GNRs. The stability assessment proved high optical stability of GNR-PEG-BBN in human blood serum up to 12h. Cytotoxicity study showed biocompatibility of GNR–PEG–BBN conjugate. Molecular targeting ability was approved in cells over expressing gastrin-releasing peptide (GRP) receptor (breast cancer cell line) in comparison with cells that do not express GRP receptor (skin fibroblast cell line). The selective accumulation of GNR-PEG-BBN was demonstrated in breast tumor in comparison with unconjugated gold nanorods, following the intravenous administration of GNR-PEG-BBN to breast tumor-bearing mice. This study demonstrated the potential of GNR-PEG-BBN as a photoacoustic imaging agent that can provide improved specificity and sensitivity for breast cancer detection.

Solid-phase proximity ligation assays for individual or parallel protein analyses with readout via real-time PCR or sequencing

Solid-phase proximity ligation assays share properties with the classical sandwich immunoassays for protein detection. The proteins captured via antibodies on solid supports are, however, detected not by single antibodies with detectable functions, but by pairs of antibodies with attached DNA strands. Upon recognition by these sets of three antibodies, pairs of DNA strands brought in proximity are joined by ligation. The ligated reporter DNA strands are then detected via methods such as real-time PCR or next-generation sequencing (NGS). We describe how to construct assays that can offer improved detection specificity by virtue of recognition by three antibodies, as well as enhanced sensitivity owing to reduced background and amplified detection. Finally, we also illustrate how the assays can be applied for parallel detection of proteins, taking advantage of the oligonucleotide ligation step to avoid background problems that might arise with multiplexing. The protocol for the singleplex solid-phase proximity ligation assay takes ~5 h. The multiplex version of the assay takes 7-8 h depending on whether quantitative PCR (qPCR) or sequencing is used as the readout. The time for the sequencing-based protocol includes the library preparation but not the actual sequencing, as times may vary based on the choice of sequencing platform.

Improved detection specificity for plasma proteins by targeting cysteine-containing peptides with photo-SRM

Targeted mass spectrometry using selected reaction monitoring (SRM) has emerged as an alternative to immunoassays for protein quantification owing to faster development time and higher multiplexing capability. However, the SRM strategy is faced with the high complexity of peptide mixtures after trypsin digestion of whole plasma or the cellular proteome that most of the time causes contamination, irremediably, by interfering compounds in the transition channels monitored. This problem becomes increasingly acute when the targeted protein is present at a low concentration. In this work, the merit of laser-induced photo-dissociation in the visible region at 473 nm implemented in an hybrid quadrupole linear ion-trap mass spectrometer (photo-SRM) was evaluated for detection specificity of cysteine-containing peptides in a group of plasma proteins after tagging with a dabcyl chromophore. Compared with conventional SRM, photo-SRM chromatograms have improved detection specificity for most of peptides monitored. Comparison of the signals obtained for the best proteotypic peptides in SRM mode and those recorded by photo-SRM of cysteine-containing peptides for the same proteins reveals either increased (up to 10-fold) or similar signal to photo-SRM detection. Finally, photo-SRM has extended response linearity across a calibration plot obtained by diluting human plasma in rat plasma, down to the lowest concentrations. Hence, photo-SRM may advantageously complement conventional SRM in assay of proteins in complex biological matrices.

Targeted protein identification, quantification and reporting for high-resolution nanoflow targeted peptide monitoring

Mass spectrometry-based targeted proteomic assays are experiencing a surge in awareness due to the diverse possibilities arising from the re-application of traditional LC-SRM technology. The FDA-approved quantitative LC-SRM-pipeline in drug discovery motivates the use to quantitatively validate putative proteomic biomarkers. However, complexity of biological specimens bears a huge challenge to identify, in parallel, specific peptides and proteins of interest from large biomarker candidate lists. Methods have been devised to increase scan speeds, improve detection specificity and verify quantitative SRM-features. In contrast, high-resolution mass spectrometers could be used to improve reliability and precision of targeted proteomics assays. Here, we present a new method for identifying, quantifying and reporting peptides in high-resolution targeted proteomics experiments performed on an orbitrap hybrid instrument using stable isotope-labeled internal reference peptides. This high precision targeted peptide monitoring (TPM) method has unique advantages over existing techniques, including the need to only detect the most abundant product ion of a given target for confident peptide identification using a scoring function that evaluates assay performance based on 1) m/z-mass accuracy, 2) retention time accuracy of observed species relative to prediction, and 3) retention time accuracy relative to internal reference peptides. Further, we show management of multiplexed precision TPM-assays using sentinel peptide standards. This article is part of a Special Issue entitled: From protein structures to clinical applications.

Leveraging bimodal kinetics to improve detection specificity

Optical microcavities are high sensitivity transducers able to detect single nanoparticles and molecules. However, the specificity of detection is dependent on the availability of an appropriate targeting moiety with minimal cross-reactivity. In the present work, an alternative approach is shown. Namely, using biotin-functionalized toroidal microcavities, the dissociation constant of biotin to two different streptavidin complexes (free and polystyrene bead) is determined. Based on the difference in affinity and in mass transport, the two complexes are identified from a mixture. By leveraging information in the binding site, improved specificity can be achieved.