Rapid and simple detection of ascorbic acid and alkaline phosphatase via controlled generation of silver nanoparticles and selective recognition.

Ascorbic acid (AA) and alkaline phosphatase (ALP) serve as an important coenzyme and enzyme in multiple biological metabolism reactions, respectively, and abnormal levels of these substrates have been associated with several diseases. Herein, a new and simple fluorescence strategy has been developed for AA and ALP sensing by exploiting CdTe quantum dots (QDs) as an effective signal indicator. This method is mainly based on the selective fluorescence-quenching reaction between Ag+ and CdTe QDs, as opposed to silver nanoparticles (Ag NPs); Ag+ can be reduced to Ag NPs by AA. Furthermore, by taking advantage of AA as a mediator, this strategy is further exploited for ALP assay given that ALP can cause the hydrolysis of l-ascorbic acid-2-phosphate (AAP), which yields AA. Under optimal conditions, controlled generation of Ag NPs and the selective recognition-based sensing system exhibit high sensitivity toward AA and ALP with limits of detection (LODs) of 3 µM and 0.25 U L-1 and linear ranges of detection from 0 to 800 µM and 1 to 1000 U L-1, respectively. Moreover, the sensor was successfully used for assaying AA in fruit juice and ALP in human serum. The results demonstrate that the proposed fluorescence strategy has significant advantages, such as its simplicity, cost-effectiveness, and rapid runtime, and the operational convenience of this label-free method further demonstrates its potential for constructing effective sensors with biochemical and clinical applications.

Sensitive CVG-AFS/ICP-MS label-free nucleic acid and protein assays based on a selective cation exchange reaction and simple filtration separation.

Nowadays, label-free atomic spectrometric bioassays are attracting great research interest because of their advantages of low cost, simple design and operation, etc. Herein, a novel and simple chemical vapor generation-atomic fluorescence spectrometry (CVG-AFS)/inductively coupled plasma-mass spectrometry (ICP-MS) label-free detection method is presented for highly sensitive and selective assay of DNA and proteins. This work mainly combined a phenomenon that CdTe quantum dots (QDs) can be used to selectively differentiate free Hg2+ and the T-Hg2+-T complex, with the use of simple membrane filtration separation to improve the performance of the label-free bioassay methods. Upon hybridization with the DNA/protein (carcinoembryonic antigen, CEA) target, the T-Hg2+-T hairpin structure was opened and Hg2+ was released; this initiated the cation exchange reaction between Hg2+ and CdTe QDs which released Cd2+ simultaneously. Subsequently, the free Cd2+ was separated by the filtration membrane without separating the CdTe QDs, which could then be separated from the sample matrices for the CVG-AFS/ICP-MS assay. Under the optimal conditions, this method possessed high sensitivity for DNA and CEA determination with limits of detection (LODs) of 0.2 nM and 0.2 ng mL-1, and linear dynamic ranges of 1-160 nM and 0.5-20 ng mL-1, respectively, and exhibited excellent DNA sequence specificity and protein selectivity. This method preserves the advantages of the label-free atomic spectrometric bioassay, and combined with the selective cation exchange reaction and simple filtration separation to improve the performance.

Phenotyping grapevine red blotch virus and grapevine leafroll-associated viruses before and after symptom expression through machine-learning analysis of hyperspectral images.

Introduction: Grapevine leafroll-associated viruses (GLRaVs) and grapevine red blotch virus (GRBV) cause substantial economic losses and concern to North America's grape and wine industries. Fast and accurate identification of these two groups of viruses is key to informing disease management strategies and limiting their spread by insect vectors in the vineyard. Hyperspectral imaging offers new opportunities for virus disease scouting. Methods: Here we used two machine learning methods, i.e., Random Forest (RF) and 3D-Convolutional Neural Network (CNN), to identify and distinguish leaves from red blotch-infected vines, leafroll-infected vines, and vines co-infected with both viruses using spatiospectral information in the visible domain (510-710nm). We captured hyperspectral images of about 500 leaves from 250 vines at two sampling times during the growing season (a pre-symptomatic stage at veraison and a symptomatic stage at mid-ripening). Concurrently, viral infections were determined in leaf petioles by polymerase chain reaction (PCR) based assays using virus-specific primers and by visual assessment of disease symptoms. Results: When binarily classifying infected vs. non-infected leaves, the CNN model reaches an overall maximum accuracy of 87% versus 82.8% for the RF model. Using the symptomatic dataset lowers the rate of false negatives. Based on a multiclass categorization of leaves, the CNN and RF models had a maximum accuracy of 77.7% and 76.9% (averaged across both healthy and infected leaf categories). Both CNN and RF outperformed visual assessment of symptoms by experts when using RGB segmented images. Interpretation of the RF data showed that the most important wavelengths were in the green, orange, and red subregions. Discussion: While differentiation between plants co-infected with GLRaVs and GRBV proved to be relatively challenging, both models showed promising accuracies across infection categories.

A general strategy for label-free homogeneous bioassays based on selective recognition and silver ion-mediated conformational switch.

Simple, sensitive, and cost-effective detection of biomolecules is important to clinical analysis and treatment. Herein, a novel fluorescence turn-off method for the homogeneous detection of DNA and proteins was developed using selective recognition reaction and silver ion-mediated conformational switch. In this work, we discovered the new phenomenon that cadmium telluride (CdTe) quantum dots (QDs) could selectively differentiate silver ions (Ag+) from the cytosine (C)-Ag+-cytosine (C-Ag+-C) hairpin structure, owing to the ability of Ag+ to quench CdTe QDs which the C-Ag+-C complex didn't possess. With this method, the C-Ag+-C hairpin probe formed, followed by the reaction with the target DNA, thus the C-Ag+-C was opened and free Ag+ were released. This initiated the selective recognition reaction with CdTe QDs, leading to the decrease in the CdTe QDs fluorescence intensity. Therefore, the DNA concentration could be quantitatively measured by indirectly monitoring the fluorescence signal of CdTe QDs. Furthermore, the application of this sensor was extended to the detection of prostate-specific antigen (PSA) by employing its aptamer as the recognize elements, with the similar strategy used for the target DNA assay. Under the optimized conditions, this strategy achieved good analytical performance for both DNA assay with a limit of detection of 0.12 nM and linear dynamic range of 5-100 nM, and PSA assay with a limit of detection of 0.01 ng/mL and the linear dynamic range of 0.1-2.5 ng/mL. This new fluorescence method is simple in design and operation, with no need for oligonucleotide labeling and separation, and it has great potential to be applied in clinical diagnosis.

Exonuclease III-assisted strand displacement reaction-driven cyclic generation of G-quadruplex strategy for homogeneous fluorescent detection of melamine.

Development of flexible, sensitive, selective and simple melamine detection is in high demand and of great significance. Here, we proposed a homogeneous turn-off mode fluorescent strategy to detect melamine by coupling the process of exonuclease III-assisted (Exo III) amplification with the strand displacement reaction (SDR)-driven assembly of DNA G-quadruplex structures. Melamine (M) could bind to the thymine (T) base through hydrogen bonding to form a T-M-T structure, which inhibits the subsequent nucleic acid amplification reaction and formation of the G-quadruplex structure. DNA strand (G1) served as both a recognition probe and a signal probe, thus greatly simplifying the experimental design and operation. Furthermore, to improve the detection sensitivity, an ingeniously designed segment of double-stranded DNA (dsDNA, P1-G2) that contained the G-quadruplex sequence (G2) and a partially hybridized signal trigger strand (P1) was added to the system. Following the SDR and Exo III-assisted amplification, G1-DNA and G2-DNA were released and the cyclic production of G-quadruplex structures was initiated. The generated DNA G-quadruplex bound to the NMM (a fluorescent dye, N-methyl mesoporphyrin IX), providing enhanced fluorescence signals. This allowed for the highly sensitive detection of melamine at concentrations as low as 25 fM. The proposed fluorescent detection method of melamine concentration displayed a good linear relationship ranging from 100 fM to 100 pM, which showed improved performance over that of the non-enzyme-assisted sensing system (limit of detection is 15 pM). Additionally, this method exhibited a high selectivity towards the target molecule, melamine, in comparison to other substances. Furthermore, the method was used for the assay of melamine in real milk samples and satisfactory experiment results were obtained. The novel strategy developed in this research may be considered as a potential route for highly sensitive, simple, selective and accurate assay of small molecules.

Active DNA unwinding and transport by a membrane-adapted helicase nanopore.

Nanoscale transport through nanopores and live-cell membranes plays a vital role in both key biological processes as well as biosensing and DNA sequencing. Active translocation of DNA through these nanopores usually needs enzyme assistance. Here we present a nanopore derived from truncated helicase E1 of bovine papillomavirus (BPV) with a lumen diameter of c.a. 1.3 nm. Cryogenic electron microscopy (cryo-EM) imaging and single channel recording confirm its insertion into planar lipid bilayer (BLM). The helicase nanopore in BLM allows the passive single-stranded DNA (ssDNA) transport and retains the helicase activity in vitro. Furthermore, we incorporate this helicase nanopore into the live cell membrane of HEK293T cells, and monitor the ssDNA delivery into the cell real-time at single molecule level. This type of nanopore is expected to provide an interesting tool to study the biophysics of biomotors in vitro, with potential applications in biosensing, drug delivery and real-time single cell analysis.

Detection of nucleic acids via G-quadruplex-controlled l-cysteine oxidation and catalyzed hairpin assembly-assisted signal amplification.

The development of simple, sensitive and cost-effective methods for specific nucleic acid detection has attracted tremendous attention due to its importance to the early diagnosis of genetic diseases and to biodefense applications. In this work, we demonstrated a fluorescent turn-off mode DNA assay based on l-cysteine-modulated synthesis of CdTe quantum dots (CdTe QDs), horseradish peroxidase-mimicking G-quadruplex-hemin-K+ complex controlled oxidation of l-cysteine to cystine, and catalyzed hairpin assembly (CHA)-assisted signal amplification. After the addition of target DNA, the CHA signal amplification reaction was triggered and numerous H1-H2 double-stranded DNA were formed, initiating the release of G-quadruplex sequences in H2 simultaneously. Thus, the degree of inhibition of the synthesis of CdTe QDs is proportional to the concentration of the G-quadruplex sequence in this method. In contrast, when the target DNA was absent, the CHA could not be triggered, and the fluorescence signal was high due to the remaining intact l-cysteine. Under optimal experimental conditions, the homogeneous fluorescence method achieved the detection of HIV DNA with a linear range from 0.1 pM to 1 nM and a detection limit of 0.12 pM. This novel biosensor exhibits excellent specificity in differentiating DNA sequences with a single-base and two-base mismatch. To the best of our knowledge, this a label-free and highly sensitive bioassay utilizing CHA-assisted signal amplification and G-quadruplex control of in situ synthesis of CdTe QDs strategy was not reported in previous. Thus, this proposed strategy is anticipated to find use in basic biochemical research and clinical diagnosis.