Robust Peptide-Functionalized Gold Nanoparticles via Ethynyl Bonding for High-Fidelity Bioanalytical Applications.

While Au-S bonds have been widely applied in preparing gold nanoparticle (AuNP) bioconjugates for biosensing, cell imaging, and biomedical research, biothiols in complex biological environments can seriously interfere with the stability of the conjugates due to ligand exchange. Herein, we communicate a robust and fast strategy for constructing peptide-functionalized AuNP conjugates (PFCs) using the Au-C≡C bond, which can be completed within two minutes. The resulting Au-C≡C PFCs exhibited better stability and resistance to biothiols than the corresponding Au-S PFCs, and also demonstrated excellent stability in high salt concentration, a wide range of pH values, and varying temperatures.

Poly(vinylpyrrolidone)-Enhanced CRISPR-Cas System for Robust Nucleic Acid Diagnostics.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has opened a new path for molecular diagnostics based on RNA programmed -cleavage activity. However, their accessibility for highly sensitive clinical diagnostics remains insufficient. In this study, we systematically investigated the impact of various surfactants on the CRISPR-Cas12a system and found that poly(vinylpyrrolidone) (PVP), a nonionic surfactant, showed the highest enhancement effect among these tested surfactants.

Universal crRNA Acylation Strategy for Robust Photo-Initiated One-Pot CRISPR-Cas12a Nucleic Acid Diagnostics.

Spatiotemporal regulation of clustered regularly interspaced short palindromic repeats (CRISPR) system is attractive for precise gene editing and accurate molecular diagnosis. Although many efforts have been made, versatile and efficient strategies to control CRISPR system are still desirable. Here, we proposed a universal and accessible acylation strategy to regulate the CRISPR-Cas12a system by efficient acylation of 2'-hydroxyls (2'-OH) on crRNA strand with photolabile agents (PLGs).

Ultrasensitive CRISPR/Cas13a-Mediated Photoelectrochemical Biosensors for Specific and Direct Assay of miRNA-21.

Sensitive and specific assay of microRNAs (miRNAs) is beneficial to early disease screening. Herein, we for the first time proposed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a-mediated photoelectrochemical biosensors for the direct assay of miRNA-21. In this study, compared with traditional nucleic acid-based signal amplification strategies, the CRISPR/Cas13a system can greatly improve the specificity and sensitivity of target determination due to its accurate recognition and high-efficient -cleavage capability without complex nucleic acid sequence design.

Development of a Handheld Nano-centrifugal Device for Visual Virus Detection.

Unlabelled: Gold nanoparticles (AuNPs) colorimetric assays based on distance-dependent optical characteristics have been widely employed for bioanalysis. However, this assay is not effective for visually detecting low-concentration targets due to the faint color change. Here, we developed a handheld nano-centrifugal device which could separate the crosslinked and non-crosslinked AuNPs.

Glycerol Additive Boosts 100-fold Sensitivity Enhancement for One-Pot RPA-CRISPR/Cas12a Assay.

CRISPR/Cas12, a highly efficient and specific nucleic acid recognition system, has been broadly employed to detect amplified DNA products. However, most reported methods adopt a two-step detection mode that needs a liquid transfer step, thus complicating the detection procedure and posing a risk of aerosol contamination. A one-pot detection method can obviate these problems, but it suffers from poor detection efficiency due to the loss of amplification templates elicited by CRISPR/Cas12 cleavage.

Developing predictive hybridization models for phosphorothioate oligonucleotides using high-resolution melting.

The ability to predict nucleic acid hybridization energies has been greatly enabling for many applications, but predictive models require painstaking experimentation, which may limit expansion to non-natural nucleic acid analogues and chemistries. We have assessed the utility of dye-based, high-resolution melting (HRM) as an alternative to UV-Vis determinations of hyperchromicity in order to more quickly acquire parameters for duplex stability prediction. The HRM-derived model for phosphodiester (PO) DNA can make comparable predictions to previously established models.

Fast microwave heating-based one-step synthesis of DNA and RNA modified gold nanoparticles.

DNA/RNA-gold nanoparticle (DNA/RNA-AuNP) nanoprobes have been widely employed for nanobiotechnology applications. Here, we discover that both thiolated and non-thiolated DNA/RNA can be efficiently attached to AuNPs to achieve high-stable spherical nucleic acid (SNA) within minutes under a domestic microwave (MW)-assisted heating-dry circumstance. Further studies show that for non-thiolated DNA/RNA the conjugation is poly (T/U) tag dependent.

Droplet Cas12a Assay Enables DNA Quantification from Unamplified Samples at the Single-Molecule Level.

DNA quantification is important for biomedical research, but the routinely used techniques rely on nucleic acid amplification which have inherent issues like cross-contamination risk and quantification bias. Here, we report a CRISPR-Cas12a-based molecular diagnostic technique for amplification-free and absolute quantification of DNA at the single-molecule level. To achieve this, we first screened out the optimal reaction parameters for high-efficient Cas12a assay, yielding over 50-fold improvement in sensitivity compared with the reported Cas12a assays.

Minimizing Leakage in Stacked Strand Exchange Amplification Circuits.

Signal amplification is ubiquitous in biology and engineering. Protein enzymes, such as DNA polymerases, can routinely achieve >10-fold signal increase, making them powerful tools for signal enhancement. Considerable signal amplification can also be achieved using nonenzymatic, cascaded nucleic acid strand exchange reactions.

Advances in Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)-Based Diagnostic Assays Assisted by Micro/Nanotechnologies.

Clustered, regularly interspaced short palindromic repeats (CRISPR)-based diagnoses, derived from gene-editing technology, have been exploited for less than 5 years and are now reaching the stage of precommercial use. CRISPR tools have some notable features, such as recognition at physiological temperature, excellent specificity, and high-efficiency signal amplification capabilities. These characteristics are promising for the development of next-generation diagnostic technologies.

A CRISPR/Cas9 eraser strategy for contamination-free PCR end-point detection.

Polymerase chain reaction (PCR), a central technology for molecular diagnostics, is highly sensitive but susceptible to the risk of false positives caused by aerosol contamination, especially when an end-point detection mode is applied. Here, we proposed a solution by designing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 eraser strategy for eliminating potential contamination amplification. The CRISPR/Cas9 engineered eraser is firstly adopted into artpcr reverse-transcription PCR (RT-PCR) system to achieve contamination-free RNA detection.

Simultaneous Dual-Gene Diagnosis of SARS-CoV-2 Based on CRISPR/Cas9-Mediated Lateral Flow Assay.

Few methods for the detection of SARS-CoV-2 currently have the capability to simultaneously detect two genes in a single test, which is a key measure to improve detection accuracy, as adopted by the gold standard RT-qPCR method. Developed here is a CRISPR/Cas9-mediated triple-line lateral flow assay (TL-LFA) combined with multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and simultaneous dual-gene detection of SARS-CoV-2 in a single strip test. This assay is characterized by the detection of envelope (E) and open reading frame 1ab (Orf1ab) genes from cell-cultured SARS-CoV-2 and SARS-CoV-2 viral RNA standards, showing a sensitivity of 100 RNA copies per reaction (25 μL).

A CRISPR-driven colorimetric code platform for highly accurate telomerase activity assay.

Telomeric repeat amplification protocol (TRAP) has been the most widely used method for assessing the telomerase activity from cells and tissues. However, cell lysates, body fluid samples, or tumor tissue samples often contain high concentrations of protein or other complex matrices, which are usually inhibiting the TRAP response, thus leading to false-negative results. Internal control (IC) involved TRAP enables reliable telomerase activity assay but requires time consuming and laborious electrophoretic separation to visualize telomeric repeat DNA and internal control products from TRAP reaction, severely limiting its application in clinical diagnosis.

CUT-LAMP: Contamination-Free Loop-Mediated Isothermal Amplification Based on the CRISPR/Cas9 Cleavage.

Loop-mediated isothermal amplification (LAMP) is a sensitive and widely used gene amplification technique. However, high amplification efficiency and amplification products containing multiple inverted repeats make the LAMP reaction extremely vulnerable to false-positive amplification caused by contamination. Herein, a contamination-free LAMP (CUT-LAMP) assisted by the CRISPR/Cas9 cleavage with superior reliability and durability has been reported.

Single-Step, Salt-Aging-Free, and Thiol-Free Freezing Construction of AuNP-Based Bioprobes for Advancing CRISPR-Based Diagnostics.

The recently reported freezing-based labeling method for constructing DNA-AuNP probes is rapid but still requires thiol modification. Here, we evaluated a poly(A)-tagged DNA sequence using the freezing-based labeling method, and the results demonstrated that approximately 10 A bases at the sequence ends are essential. More detailed observations revealed that some DNA sequences tend to form secondary structures and thus shield exposed A bases, resulting in inefficient or failed labeling.

Dynamic Programming of a DNA Walker Controlled by Protons.

We have now constructed a four-legged DNA walker based on toehold exchange reactions whose movement is controlled by alternating pH changes. A well-characterized, pH-responsive CG-C triplex DNA was embedded into a tetrameric catalytic hairpin assembly (CHA) walker. The proton-controlled walker could autonomously move on otherwise unprogrammed microparticles surface, and the walking rate and steps of walking were efficiently controlled by pH.

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Mediated Lateral Flow Nucleic Acid Assay.

The lateral flow assay is one of the most convenient analytical techniques for analyzing the immune response, but its applicability to precise genetic analyses is limited by the false-positive signal and tedious and inefficient hybridization steps. Here, we introduce the CRISPR (clustered regularly interspaced short palindromic repeats) /Cas system into the lateral flow assay, termed CRISPR/Cas9-mediated lateral flow nucleic acid assay (CASLFA), to address such issues. In this study, CASLFA is utilized to identify , genetically modified organisms (GMOs), and African swine fever virus (ASFV) at a detection limit of hundreds of copies of genome samples with high specificity within 1 h.

Universal and Naked-Eye Gene Detection Platform Based on the Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a/13a System.

Gold-nanoparticles-based colorimetric assay is an attractive detection format, but is limited by the tedious and ineffective posthybridization manipulations for genomic analysis. Here, we present a new design for a colorimetric gene-sensing platform based on the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system. In this strategy, programmable recognition of DNA by Cas12a/crRNA and RNA by Cas13a/crRNA with a complementary target activates the -ssDNA or -ssRNA cleavage.

CRISPR/Cas13a Signal Amplification Linked Immunosorbent Assay for Femtomolar Protein Detection.

The enzyme-linked immunosorbent assay (ELISA) is a basic technique used in analytical and clinical investigations. However, conventional ELISA is still not sensitive enough to detect ultralow concentrations of biomarkers for the early diagnosis of cancer, cardiovascular risk, neurological disorders, and infectious diseases. Herein we show a mechanism utilizing the CRISPR/Cas13a-based signal export amplification strategy, which double-amplifies the output signal by T7 RNA polymerase transcription and CRISPR/Cas13a collateral cleavage activity.

Binding-Induced 3D-Bipedal DNA Walker for Cascade Signal Amplification Detection of Thrombin Combined with Catalytic Hairpin Assembly Strategy.

As an important biomarker, thrombin (TB) is a major player in thrombosis and hemostasis and has attracted increasing attention involving its determination. Herein a universal and ultrasensitive fluorescence biosensor based on a binding-induced 3D-bipedal DNA walker and catalytic hairpin assembly (CHA) strategy has been proposed for cascade signal amplification detection of thrombin. In this study, we designed two proximity probes (foot 1 and foot 2) which include a specific affinity ligand for TB binding and a Pb-dependent DNAzyme tail sequence.

Nd@TiO₂ Nanocomposite for Photocatalytic Inactivation of .

In present work, a novel Nd@TiO₂ Nanocomposite, synthesized successfully by a facile sol-gel method, reveals significant light-activated antibacterial activity. The X-ray diffraction (XRD), Raman spectroscopy and transmission electron microscopy (TEM) show the anatase phase and globular shape of Nd@TiO₂. UV-vis diffuse reflectance spectroscopy and low temperature N₂ adsorption (BET) indicate Nd@TiO₂ has the narrow band gap (3.

Cascade signal amplified assay of nucleic acids based on entropy-driven amplification strategy and Mg-dependent DNAzyme cleavage.

Nucleic acids-based biosensors are extremely important in modern life sciences and have been widely used for the detection of many biomarkers of disease and extensively applied in many fields, such as medical analysis, gene therapy, and pathogen determination. Therefore, it is necessary to develop some sensitive and selective methods for rapid detection of nucleic acids. In this work, an ultrasensitive and non-enzyme electrochemical biosensor has been developed for nucleic acids detection based on entropy-driven amplification (EDA) strategy and Mg-dependent DNAzyme cleavage method.

An ultrasensitive electrochemical immunoassay based on a proximity hybridization-triggered three-layer cascade signal amplification strategy.

In this paper, an ultrasensitive electrochemical immunoassay was constructed for the determination of carcinoembryonic antigen (CEA) based on a proximity hybridization-triggered three-layer cascade signal amplification strategy. In the presence of CEA and two antibody-labeled DNA strands (Ab-DNA1 and Ab-DNA2), a sandwich proximate complex was formed which could hybridize with the hairpin DNA (HP1) and open it, initiating the degradation process of exonuclease III (Exo III) accompanied by the release of a sandwich proximate complex used for Cycle I and autogenous yield of a DNA fragment. The fragment was then employed as a ST-DNA (secondary target DNA fragment) for the subsequent binding to the other hairpin DNA (HP2) on the gold (Au) electrode surface to trigger the catalytic hairpin assembly (CHA, Cycle II) and rolling circle amplification (RCA).