Designing a fluorescence padlock probe-based biosensor and colorimetric assay for the detection of G12D KRAS mutation

Aim: Cell-free DNA in the plasma is known to be a potential biomarker for noninvasive diagnosis of oncogenic mutations. The authors aimed to design an optimized padlock probe-based hyperbranched rolling circle amplification biosensor to detect the KRAS G12D mutation using fluorescence and colorimetric methods. Methods: Single-factor experiments, Plackett–Burman design and response surface methodology were applied to optimize the padlock probe-based hyperbranched rolling circle amplification reaction. Results: The maximum fluorescence intensity was achieved at a padlock probe concentration of 1.5 pM and target concentration of 9 pM at 38°C ligation temperature. The proposed biosensor has a low detection limit of 60 fM of target DNA and a linear response in the concentration range of 60 fM to 0.2 pM. Conclusion: The results indicated the power of these assays to detect KRAS point mutations in liquid state reactions.

Image Processing and Luminescent Probes for Bioimaging Techniques with High Spatial Resolution and High Sensitivity

The balance of microenvironmental factors (including temperature, pH, ROS species, etc.) plays a crucial role in maintaining normal living organisms’ normal physiological activities and physiological functions. Therefore, armed with the unique superiorities of high spatial resolution, non-invasion, high sensitivity, real-time monitoring, and simple operation, luminescent imaging technology has been widely used in real-time and accurate monitoring of microenvironmental factors in these organisms to prevent, diagnose and treat related diseases in time. However, due to its optical imaging characteristics, it is also faced with such interference factors as relatively shallow imaging penetration depth, background fluorescence (biological autofluorescence) interference in a complex environment, uncertain probe concentration, and unstable laser power in the imaging process, which are not related to the analyte. As for the problems in imaging, such as the uncertainty of probe concentration and the fluctuation of instrument laser power, the ratio detection, and imaging technology with self-calibration function can effectively avoid these problems. As for background fluorescence interference in imaging, probes with long-life emission can be used in imaging. The long-life luminescence of probes from background fluorescence can be recognized by time-resolved luminescence imaging technology to reduce its impact. This paper briefly introduces and summarizes the relative research of ratio detection and imaging technology and time-resolved luminescence imaging technology.

Noval Dual-Emission Fluorescence Carbon Dots as a Ratiometric Probe for Cu2+ and ClO− Detection

The use of carbon dots (CDs) with dual emission based on ratiometric fluorescence has been attracting attention in recent times for more accurate ion detection since they help avoid interference from background noise, probe concentration, and complexity. Herein, novel dual-emission nitrogen-doped CDs (NCDs) were prepared by a simple method for Cu2+ and ClO- detection. The NCDs showed excellent anti-interference ability and selectivity for different emissions. In addition, a good linear relationship was observed between the fluorescence intensity (FI) of the NCD solutions in different emissions with Cu2+ (0–90 μM) and ClO- (0–75 μM). The limits of both Cu2+ detection and ClO- were very low, at 17.7 and 11.6 nM, respectively. The NCDs developed herein also showed a good recovery rate in water for Cu2+ and ClO− detection. Hence, they are expected to have a more extensive application prospect in real samples.

Overcoming Supply Shortage for SARS-CoV-2 Detection by RT-qPCR

In February 2020, our laboratory started to offer a RT-qPCR assay for the qualitative detection of severe acute respiratory syndrome coronavirus 2. A few months after the assay was released to our patients, some materials, reagents, and equipment became in short supply. Alternative protocols were necessary in order to avoid stopping testing to the population. However, the suitability of these alternatives needs to be validated before their use. Here, we investigated if saliva is a reliable alternative specimen to nasopharyngeal swabs; if 0.45% saline is a reliable alternative to guanidine hydrochloride as a collection viral transport media; the stability of SARS-COV-2 in guanidine hydrochloride and in 0.45% saline for 10 and 50 days at room temperature; and if the primers/probe concentration and thermocycling times could be reduced so as to overcome the short supply of these reagents and equipment, without a significant loss of the assay performance. We found that saliva is not an appropriated specimen for our method—nasopharyngeal swabs perform better. Saline (0.45%) and guanidine hydrochloride have a similar SARS-CoV-2 diagnostic capability as tube additives. Reliable SARS-CoV-2 RNA detection can be performed after sample storage for 10 days at room temperature (18–23 °C) in both 0.45% saline and guanidine hydrochloride. Using synthetic RNA, and decreasing the concentration of primers by five-fold and probes by 2.5-fold, changed the assay limit of detection (LOD) from 7.2 copies/reaction to 23.7 copies/reaction and the subsequent reducing of thermocycling times changed the assay LOD from 23.7 copies/reaction to 44.2 copies/reaction. However, using real clinical samples with Cq values ranging from ~12.15 to ~36.46, the results of the three tested conditions were almost identical. These alterations will not affect the vast majority of diagnostics and increase the daily testing capability in 30% and increase primers and probe stocks in 500% and 250%, respectively. Taken together, the alternative protocols described here overcome the short supply of tubes, reagents and equipment during the SARS-CoV-2 pandemic, avoiding the collapse of test offering for the population: 105,757 samples were processed, and 25,156 SARS-CoV-2 diagnostics were performed from 9 May 2020 to 30 June 2020.

Quantitatively Monitoring in situ Mitochondrial Thermal Dynamics by Upconversion Nanoparticles

Temperature dynamics reflect the physiological conditions of cells and organisms. Mitochondria regulates temperature dynamics in living cells, as they oxidize the respiratory substrates and synthesize ATP, with heat being released as a by-product of active metabolism. Here, we report an upconversion nanoparticles based thermometer that allows in situ thermal dynamics monitoring of mitochondria in living cells. We demonstrate that the upconversion nanothermometers can efficiently target mitochondria and the temperature responsive feature is independent of probe concentration and medium conditions. The relative sensing sensitivity of 3.2% K−1 in HeLa cells allows us to measure the mitochondrial temperature difference through the stimulations of high glucose, lipid, Ca2+ shock and the inhibitor of oxidative phosphorylation. Moreover, cells display distinct response time and thermal dynamic profiles under different stimulations, which highlights the potential applications of this thermometer to study in situ vital processes related to mitochondrial metabolism pathways and interactions between organelles.

Direct digital sensing of proteins in solution through single-molecule optofluidics

Highly sensitive detection of proteins is of central importance to biomolecular analysis and diagnostics. Conventional protein sensing assays, such as ELISAs, remain reliant on surface-immobilization of target molecules and multi-step washing protocols for the removal of unbound affinity reagents. These features constrain parameter space in assay design, resulting in fundamental limitations due to the underlying thermodynamics and kinetics of the immunoprobe–analyte interaction. Here, we present a new experimental paradigm for the quantitation of protein analytes through the implementation of an immunosensor assay that operates fully in solution and realizes rapid removal of excess probe prior to detection without the need of washing steps. Our single-step optofluidic approach, termed digital immunosensor assay (DigitISA), is based on microfluidic electrophoretic separation combined with single-molecule laser-induced fluorescence microscopy and enables calibration-free in-solution protein detection and quantification within seconds. Crucially, the solution-based nature of our assay and the resultant possibility to use arbitrarily high probe concentrations combined with its fast operation timescale enables quantitative binding of analyte molecules regardless of the capture probe affinity, opening up the possibility to use relatively weak-binding affinity reagents such as aptamers. We establish and validate the DigitISA platform by probing a biomolecular biotin–streptavidin binding complex and demonstrate its applicability to biomedical analysis by quantifying IgE–aptamer binding. We further use DigitISA to detect the presence of α-synuclein fibrils, a biomarker for Parkinson’s disease, using a low-affinity aptamer at high probe concentration. Taken together, DigitISA presents a fundamentally new route to surface-free specificity, increased sensitivity, and reduced complexity in state-of-the-art protein detection and biomedical analysis.

Development and Evaluation of a Single Dye Duplex Droplet Digital PCR Assay for the Rapid Detection and Quantification of Mycobacterium tuberculosis

Droplet digital PCR (ddPCR) is a third generation of PCR that was recently developed to overcome the challenges of real-time fluorescence-based quantitative PCR (qPCR) in absolute quantification of pathogens. Few studies have been done on tuberculosis (TB) detection and quantification using ddPCR despite its many advantages over qPCR. From the few studies, none explores a single dye duplex assay for the detection and quantification of TB. In this study, steps toward developing and evaluating a duplex single dye (FAM) assay for detecting two targets (IS6110 and IS1081) are clearly described using simplex and duplex experiments. To achieve this, various parameters are investigated, including annealing temperature, primer and probe concentration, sensitivity and specificity, sample concentration, and inter/intra-assay variability. From the results, primer and probe concentration, annealing temperature, and sample concentration have an effect on the position and separation of droplets in both simplex and duplex assays. The copies of target genes in a duplex assay can be estimated accurately using the threshold tool with little inter-assay (CV <1%) and intra-assay (CV <6%) variability when compared to simplex assays. The ddPCR assay specificity and sensitivity are both 100% when compared to qPCR. This work shows steps toward the detection and quantification of two targets in a single channel, enabling higher multiplexing to include more targets in future works.

Preparation and Application of Multi-functional His-AA-AuNCs Fluorescent Probe

Functional nanomaterials with simulated properties have become promising candidates for the detection of hydrogen peroxide. However, there are few studies on the colorimetric detection of metal ions and amino acids based on the peroxidase-simulation activity of amino acid functionalized AuNCs. In this study, a method for preparing fluorescent probe using histidine (His) and ascorbic acid (AA) as reductant and stabilizer was proposed. TMB was used as chromogenic substrate to indicate the catalytic process. The ultraviolet absorbance of oxTMB was determined at the characteristic absorption peak 625nm, and the standard curve was drawn to determine the concentration of H2O2 accurately. We found that Fe3+ can greatly improve the response signal of His-AA-AuNCs and has high selectivity. The linear range of H2O2 concentration detection is 10-9.97×106 μM. The concentration range of probe response to Fe3+ is 0.28-280 nM. The His-AA-AuNCs fluorescent probe was applied to intracellular fluorescence imaging after adriamycin injury, and the fluorescence intensity increased with the increase of probe concentration. This study was based on the double-stranded nature of amino acids and the properties of hydrogen peroxide mimic enzymes to detect other substances, which has a promising application prospect, and may potentially be applied to metal ions, amino acids and peptides in the biological and environmental fields in the future.