Reporter Probe Research Articles

Detection of Vibrio harveyi through RPA combined with CRISPR/Cas12a: Construction of a new method and comparative study

In this study, recombinase polymerase isothermal amplification (RPA) was combined with clustered regularly interspaced short palindromic repeats/associated proteins (CRISPR/Cas) to establish a new method (RPA-CRISPR/Cas12a) for the detection of Vibrio harveyi. We used RPA to amplify the target DNA of V. harveyi and the RPA amplification product as an activator to initiate CRISPR/Cas12a cleavage of the designed ssDNA reporter probe. Then, we used fluorescence intensity as a criterion for the detection results to optimize the reaction conditions and improve the accuracy of the assay. The ToxR-specific gene sequence of V. harveyi was selected, and 3 sets of RPA primers and 2 sets of gRNA were designed for screening to optimize the ratio of LbCas12a protein, gRNA, and reporter. The specificity, sensitivity and detection of the newly developed method on simulated contaminated samples were analyzed and compared with those of the RPA method, fluorescent loop mediated isothermal amplification (LAMP), and visual LAMP methods. The results showed that the newly constructed method exhibited good specificity and no cross-reactivity with other pathogenic bacteria. Maximum fluorescence values were obtained when the molar concentration ratios of LbCas12a protein to reporter and gRNA were 1:0.8 and 1:8, respectively. The sensitivities of RPA-CRISPR/Cas12a for the detection of V. harveyi were 10−5 ng/μL, respectively, and the sensitivities for the simulated contaminated samples were 7 CFU/mL, respectively. The new RPA-CRISPR/Cas12a method provides a new strategy for the immediate detection of foodborne pathogenic microorganisms.

Toxicity of nuclear-localized GFP in reporter mice

Various techniques using fluorescent reporter probes have been developed, such as GFP transgenic mouse lines that are used to detect spatial–temporal expression levels of genes. Although GFP expression is largely considered non-toxic, recent reports have indicated that under certain conditions GFP can display cellular toxicity. We hereby report the nuclear toxicity of H2B-GFP using a K14 specific Tet-on reporter mouse system. Using this system, GFP accumulates in the nucleus of all K14 expressing cells, such as the ocular surface epithelia and ocular adnexa. Expression of high levels of nuclear GFP during embryonic stages led to an eye open-at-birth (EOB) phenotype and abnormal ocular adnexa development and during adult and aging stages showed notable toxicity to ocular tissues. Other tissues, such as skin, also presented multiple defects associated with H2B-GFP expression. This toxicity was found to be concentration dependent, with homozygous mice presenting extremely high toxicity, while heterozygous mice presented limited toxicity. Upon induction, the accumulation of H2B-GFP in the nucleus of homozygous mice led to apoptosis within 2 weeks. This study therefore shows that although the use of nuclear GFP reporter mice is a valuable tool, at high levels, nuclear GFP can be toxic, leading to cell death and affecting tissue function.

Highly sensitive and specific electrochemical biosensor for direct detection of hepatitis C virus RNA in clinical samples using DNA strand displacement

Hepatitis C virus (HCV) is a common blood-borne infection that can lead to long-term illnesses such as hepatocellular cancer and liver cirrhosis. Early diagnosis is crucial for effective management, as no vaccine is available for preventing HCV infection. However, the high cost and complexity of current molecular diagnostic tools hinder efforts to achieve early diagnosis and prevent transmission, particularly in resource-limited settings. We developed a novel electrochemical biosensor for point-of-care testing (POCT) of HCV RNA. The sensor utilizes a strand displacement method, where the target RNA displaces a gold nanoparticle-labeled reporter probe (AuRP) from a pre-hybridized duplex with a magnetic nanoparticle (MNP)-labeled capture probe. The amount of displaced AuRP, detected using differential pulse anodic stripping voltammetry (DPASV), is directly proportional to the target RNA concentration. The biosensor exhibited excellent analytical performance, with a detection limit of 4 fM for synthetic targets and 43 ng/µL for RT-PCR products. Importantly, it successfully detected HCV RNA directly in clinical plasma samples without the need for RNA extraction or amplification. The sensor was used to analyze 30 RNA samples from HCV-positive patients, 20 cDNA samples from viral RNA, 30 HCV-positive plasma samples, and 22 HCV-negative plasma samples. The sensor results show good concordance with the RT-PCR results, demonstrating the sensor’s potential for detecting HCV in clinical samples.

A novel CRISPR/Cas13a biosensing platform comprising dual hairpin probe and traditional lateral flow assays

Recently, CRISPR/Cas13a-based biosensors have been combined with non-traditional lateral flow assays (N-TLFAs) successfully used to clinical detection of target RNA due to their simplicity, portability, and cost-effectiveness. However, N-TLFAs used in such biosensors have several disadvantages, such as inversion of test and control zones, in which color intensity depends on reporter probe cleavage. Herein, we propose a novel biosensor in which CRISPR/Cas13a is integrated with traditional lateral flow assays (TLFAs) to facilitate the sequential direct location of test/control zones by dual hairpin probes, namely DNA hairpin reporter probe B-HPs (5′ Biotin-hairpins, B-HPs, loop containing 5 rU) and F-HPs (5′ FAM-hairpins, F-HPs). Cas13a activated by crRNA-targeted RNA duplexes can indiscriminately cleave B-HPs reporter probes to release short sequences to open hairpin F-HPs reporter probes to form Biotin-dsDNA-FAM reporter probes. The advantage of this platform is that detection results only rely on the formed Biotin-dsDNA-FAM reporter probes, which can be used for SARS-CoV-2 RNA detection after preliminary detection. Under optimized conditions, the SARS-CoV-2 RNA detection range was 10 aM to 1 µM and the limit of detection was as low as 5.34 aM. This proof-of-concept study demonstrates that dual-hairpin reporter-probe binding to TLFAs opens up new opportunities for CRISPR/Cas13a activity detection and bioanalytical applications.

Microdroplet-SERS platform for single cell-secreted VEGF and extracellular pH analysis in oxidative stress event

Although oxidative stress processes based on reactive oxygen species have been the research focus, analyzing this phenomenon at the single-cell level is challenging. A microfluidic platform combined with highly sensitive surface-enhanced Raman scattering (SERS) technology was constructed. The secretion of the vascular endothelial growth factor (VEGF) and the extracellular microenvironmental pH fluctuations of a single cell during the oxidative stress process were assessed and analyzed. The immune sandwich structure formed between the capture probe and the reporter probe with the linkage of secreted VEGF was built above the probed cell surface in each drop. The self-driven collection behavior of the magnetic bead-based capture probe toward the cell surface significantly amplified the SERS signals of the reporter probe, thus improving the sensitivity and accuracy of detection. 4-Mercaptopyridine (4-Mpy) responding sensitively to pH was applied to label the reporter probe, which endows the extracellular microenvironmental pH sensing during oxidative stress events according to relative SERS intensity. Combined with machine learning to analyze the spectral characteristics, the distinction between normal cells and different types of tumor cells was finally realized. This study makes full use of the advantages of machine learning to process spectral data to explore deep information and provides an auxiliary tool for diagnosing cancer and other medical conditions in the future.

An electrochemical biosensor based on MoS2-PANi@Fc for the detection of Golgi protein 73

Golgi protein 73 (GP73) is highly expressed in hepatocellular carcinoma (HCC) and has been regarded as a sensitive serum biomarker for HCC diagnosis. Herein, an electrochemical biosensor for the determination of GP73 was constructed based on the molybdenum disulfide-polyaniline@ferrocene (MoS2-PANi@Fc) nanocomposites. The MoS2-PANi@Fc was prepared with large surface area and excellent electrical properties. One aptamer for GP73 (GP73Apt1) immobilized on MoS2-PANi@Fc by π-π stacking and metal ligand interactions was used as an electrochemical signal reporter probe by Fc oxidation. Another GP73 aptamer (GP73Apt2) was immobilized on the surface of gold nanoparticles electrodeposition into screen-printed carbon electrode (Au NPs/SPCE) to capture the target GP73. In the presence of GP73, Fc electrochemical signal recorded by differential pulse voltammetry (DPV) increased gradually. The preparation processes, mechanisms and optimal experiment conditions of electrochemical biosensor were studied by electron microscope imaging, spectral curves and electrochemical measurements. Under optimized experimental conditions, the aptasensor was demonstrated to be capable of detecting GP73 with a linear range of 0.001–100.0 ng mL−1 and a limit of detection (LOD) of 0.001 ng mL−1 (σ = 3). Stability, specificity and reproducibility were well demonstrated. These results may provide new insights for clinical detection of HCC.

In-droplet multiplex immunoassays for hypoxia-induced single-cell cytokines

Cytokine expression is an important biomarker in understanding hypoxia microenvironments in tumor growth and metastasis. In-droplet-based immunoassays performed above the target cell membrane were employed to track the cytokines of single cells with the aid of three types of immuno-nanoprobes (one capture nanoprobe and two reporter nanoprobes). Single cells and nanoprobes were co-packaged in water-in-oil microdroplets (about 100 μm in diameter) using a cross-shaped microfluidic chip. In each droplet, capture nanoprobes would be first fixed to the cell surface by linking to membrane proteins that have been streptavidinized. Then, the capture nanoprobes can collect cell-secreted cytokines (VEGF and IL-8) by the antibodies, followed by two reporter nanoprobes that emit distinguishable fluorescence. Fluorescence imaging was utilized to record the signal outputs of two reporter probes, which reflect cytokine expressions secreted by a single tumor cell. The cytokine levels at different degrees of hypoxia induction were assessed. Multiple chemometric methods were adopted to distinguish differences in the secretion of two cytokines and the results demonstrated a positive correlation. This study developed an in-droplet, dual-target, simultaneous biosensing strategy for a single cell, which is helpful for understanding the impacts of hypoxia microenvironments on cell cytokines that are vital for assessing early cancer diagnosis and prognosis.

Rapid detection of pathogenic E. coli based on CRISPR Cas system.

Access to safe and nutritious food is critical for maintaining life and supporting good health. Eating food that is contaminated with pathogens leads to serious diseases ranging from diarrhea to cancer. Many foodborne infections can cause long-term impairment or even death. Hence, early detection of foodborne pathogens such as pathogenic Escherichia coli strains is essential for public safety. Conventional methods for detecting these bacteria are based on culturing on selective media and following standard biochemical identification. Despite their accuracy, these methods are time-consuming. PCR-based detection of pathogens relies on sophisticated equipment and specialized technicians which are difficult to find in areas with limited resources. Whereas CRISPR technology is more specific and sensitive for identifying pathogenic bacteria because it employs programmable CRISPR-Cas systems that target particular DNA sequences, minimizing non-specific binding and cross-reactivity. In this project, a robust detection method based on CRISPR-Cas12a sensing was developed, which is rapid, sensitive and specific for detection of pathogenic E. coli isolates that were collected from the fecal samples from adult goats from 17 farms in Tennessee. Detection reaction contained amplified PCR products for the pathogenic regions, reporter probe, Cas12a enzyme, and crRNA specific to three pathogenic genes-stx1, stx2, and hlyA. The CRISPR reaction with the pathogenic bacteria emitted fluorescence when excited under UV light. To evaluate the detection sensitivity and specificity of this assay, its results were compared with PCR based detection assay. Both methods resulted in similar results for the same samples. This technique is very precise, highly sensitive, quick, cost effective, and easy to use, and can easily overcome the limitations of the present detection methods. This project can result in a versatile detection method that is easily adaptable for rapid response in the detection and surveillance of diseases that pose large-scale biosecurity threats to human health, and plant and animal production.

A programmable sensitive platform for pathogen detection based on CRISPR/Cas12a -hybridization chain reaction-poly T-Cu

Rapid and sensitive detection of pathogenic bacteria is crucial for disease prevention and control. The CRISPR/Cas12a system with the DNA cleavage capability holds promise in pathogenic bacteria diagnosis. However, the sensitivity of CRISPR-based assays remains a challenge. Herein, we report a versatile and sensitive pathogen sensing platform (HTCas12a) based on the CRISPR/Cas12a system, hybridization chain reaction (HCR) and Poly T-copper fluorescence nanoprobe. The sensitivity is improved by HCR and the Poly-T-Cu reporter probe reduces the overall experiment cost to less than one dollar per sample. Our results demonstrate the specific recognition of target nucleic acid fragments from other pathogens. Furthermore, a good linear correlation between fluorescence intensity and target quantities were achieved with detection limits of 23.36 fM for Target DNA and 4.17 CFU/mL for S.aureus, respectively. The HTCas12a system offers a universal platform for pathogen detection in various fields, including environmental monitoring, clinical diagnosis, and food safety.

Exploration of new ways for CRISPR/Cas12a activation: DNA hairpins without PAM and toehold and single strands containing DNA and RNA bases

The CRISPR/Cas12a system is emerging as a promising candidate for next-generation diagnostic biosensing platforms, with the discovery of new activation modes greatly expanding its applications. Here, we have identified two novel CRISPR/Cas12a system activation modes: PAM- and toehold-free DNA hairpins, and DNA-RNA hybrid strands. Utilizing a well-established real-time fluorescence method, we have demonstrated a strong correlation between DNA hairpin structures and Cas12a activation. Compared with previously reported activation modes involving single-stranded DNA and PAM-contained double-stranded DNA, the DNA hairpin activation way exhibits similar specificity and generality. Moreover, our findings indicate that increasing the number of RNA bases in DNA-RNA hybrid strands can decelerate the kinetics of Cas12a-triggered trans-cleavage of reporter probes. These newly discovered CRISPR/Cas12a activation ways hold significant potential for the development of high-performance biosensing strategies.

Ultrasensitive SERS aptasensor using Au@Ag bimetallic nanorod SERS tags for the selective detection of amantadine in foods

Herein, a novel surface enhanced Raman spectroscopy (SERS) aptasensor was developed for amantadine (AMD) detection, based on magnetite nanoparticles coated with polyethylenimine, silver nanoclusters and aptamers (Fe3O4@PEI@AgNC-apt) as the capture probe and complementary DNA-modified gold nanorods (AuNRs@4-MPBA@Ag-c-DNA containing 4-mercaptophenylboric acid molecules) as the reporter probe. In the presence of AMD, the AMD and the reporter probe competed for the aptamer on the surface of the capture probe, resulting in the reporter probe detaching from the capture probe leading to a decrease in intensity of the SERS signal at 1067 cm−1 for 4-MPBA. Under optimal conditions, a good linear relationship was established between the SERS intensity at 1067 cm−1 and the logarithm of the AMD concentration over the range 10−6-102 mg L−1, with a LOD of 0.50 × 10−6 mg L−1. The AMD levels in spiked samples were evaluated using the SERS aptasensor, with good recoveries ranging from 90.57% to 113.49% being obtained.

Soft interface confined DNA walker for sensitive and specific detection of SARS-CoV-2 variants

Nucleic acid detection is conducive to preventing the spread of COVID-19 pandemic. In this work, we successfully designed a soft interface confined DNA walker by anchoring hairpin reporter probes on cell membranes for the detection of SARS-CoV-2 variants. In the presence of target RNA, the cyclic self-assembly reaction occurred between hairpin probes H1 and H2, and the continuous walking of target RNA on cell membranes led to the gradual amplification of fluorescence signal. The enrichment of H1 on membranes and the unique fluidity of membranes promoted the collision efficiency between DNA strands in the reaction process, endowing this method with high sensitivity. In addition, the double-blind test of synthetic RNA in 5% normal human serum demonstrated the good stability and anti-interference in complex environment of this method, which exhibited great potential in clinical diagnostics.

Exploring the prognostic value of circular RNAs in pancreatic ductal adenocarcinoma using genome-wide expression profiling

Background/ObjectivesMedian survival of pancreatic ductal adenocarcinoma (PDAC) is around eight months and new prognostic tools are needed. Circular RNAs (circRNAs) have gained interest in different types of cancer. However, only a few studies have evaluated their potential in PDAC. We aimed to identify the most differentially expressed circRNAs in PDAC compared to controls and to explore their potential as prognostic markers. MethodsUsing frozen specimens with PDAC and controls, we performed RNA sequencing and identified 20,440 unique circRNAs. A custom code set of capture- and reporter probes for NanoString nCounter analysis was designed to target 152 circRNAs, based on abundancy, differential expression and a literature study. Expression of these 152 circRNAs was examined in 108 formalin-fixed and paraffin-embedded surgical PDAC specimens and controls. The spatial expression of one of the most promising candidates, ciRS-7 (hsa_circ_0001946), was evaluated by chromogenic in situ hybridization (CISH) using multi-punch tissue microarrays (TMAs) and digital imaging analysis. ResultsBased on circRNA expression profiles, we identified different PDAC subclusters. The 30 most differentially expressed circRNAs showed log2 fold changes from −3.43 to 0.94, where circNRIP1 (hsa_circ_0004771), circMBOAT2 (hsa_circ_0007334) and circRUNX1 (hsa_circ_0002360) held significant prognostic value in multivariate analysis. CiRS-7 was absent in PDAC cells but highly expressed in the tumor microenvironment. ConclusionsWe identified several new circRNAs with biomarker potential in surgically treated PDAC, three of which showed an independent prognostic value. We also found that ciRS-7 is absent in cancer cells but abundant in tumor microenvironment and may hold potential as marker of activated stroma.

A dual-core 3D DNA nanomachine based on DNAzyme positive feedback loop for highly sensitive MicroRNA imaging in living cells

A double 3D DNA walker nanomachine by DNAzyme self-driven positive feedback loop amplification for the detection of miRNA was constructed. This method uses two gold nanoparticles as the reaction core, and because of the spatial confinement effect the local concentration of the reactants increase the collision efficiency was greatly improved. Meanwhile, the introduction of positive feedback loop promotes the conversion efficiency. In presence of miRNA-21, a large amount of DNAzyme was released and hydrolyze the reporter probe, resulting the recovery of fluorescence signal. The linear range for miRNA-21 is 0.5–60 pmol/L, and the detection limit is 0.41 pmol/L (S/N = 3). This nanomachine has been successfully used for accurate detection of miRNA-21 expression levels in cell lysates. At the same time, it can enter cells for intracellular miRNA-21 fluorescence imaging, distinguishing tumor cells from normal cells. This combination of in vitro detection and imaging analysis of living cells can achieve the goal of jointly detecting cancer markers through multiple pathways, providing new ideas for early diagnosis and screening of diseases.

Dark-field imaging and fluorescence dual-mode detection of microRNA-21 in living cells by core-satellite plasmonic nanoprobes

The in situ precise quantification and simultaneous imaging of low abundance microRNAs (miRNAs) within living cells is critical for cancer diagnosis, yet it remains a significant challenge. Leveraging the excellent sensitivity and spatiotemporal resolution of dark-field microscopy (DFM) and fluorescence imaging, we have successfully devised a novel detection approach using dual-signal reporter probes (DSRPs). These probes allow for highly sensitive detection of miRNA-21 in living cells via toehold-mediated strand displacement cascades. The DSRPs were constructed by Au nanoparticles and Ag nanoclusters core-satellite nanostructures. After the recognition of miRNA-21, the strand displacement cascades were triggered, inducing the disassembly of the Au/Ag core-satellite nanostructure with apparent scattering intensity decrease and peak wavelength shifts. Additionally, the fluorescence of Ag clusters could be recovered and further enhanced when in close proximity to specific guanine-rich strands. The dual-signal response capability enables the accurate detection of miRNA-21 from 1 fM to 1 nM, with a limit of detection reached 0.75 fM. DFM and fluorescent imaging of living cells efficiently confirms the applicable detection of miRNA-21 in complex detection media. The biosensor based on DSRPs represents a promising nanoplatform for visual monitoring and imaging of biomolecules in living cells, even at the single particle level.

Sandwich-type electrochemical aptasensor based on hemin-graphite oxide as a signal label and rGO/MWCNTs/chitosan/carbon quantum dot modified electrode for sensitive detection of Acinetobacter baumannii bacteria

Acinetobacter baumannii (A. baumannii) is a pathogenic bacterium that causes severe infections and its rapid and reliable diagnosis is essential for effective control and treatment. In this study, we present an electrochemical aptasensor based on a signal amplification strategy for the detection of A. baumannii, the high specificity and affinity of the aptamer for the target make it favorable for signal amplification. This allows for a highly sensitive and selective detection of the target. The aptasensor is based on a carbon screen-printed electrode (CSPE) that has been modified with a nanocomposite consisting of multi-walled carbon nanotubes (MWCNTs), reduced graphene oxide (rGO), chitosan (CS), and a synthesized carbon quantum dot (CQD) from CS. Additionally, the self-assembled aptamers were immobilized on hemin-graphite oxide (H-GO) as a signal probe.The composition of the nanocomposite (rGO-MWCNT/CS/CQD) provides high conductivity and stability, facilitating the efficient capture of A. baumannii onto the surface of the aptasensor. Also, aptamer immobilized on Hemin-graphite oxide (H-GO/Aptamer) was utilized as an electrochemical signal reporter probe by H reduction. This approach improved the detection sensitivity and the aptamer surface density for detecting A. baumannii. Furthermore, under optimized experimental conditions, the aptasensor was demonstrated to be capable of detecting A. baumannii with a linear range of (10 – 1 × 107 Colony-forming unit (CFU)/mL) and a limit of detection (LOD) of 1 CFU/mL (σ = 3). One of the key features of this aptasensor is its ability to distinguish between live and dead bacteria cells, which is very important and critical for clinical applications. In addition, we have successfully detected A. baumannii bacteria in healthy human serum and skim milk powder samples provided using the prepared electrochemical aptasensor.The functional groups present in the synthetic CQD, rGO-MWCNT, and chitosan facilitate biomolecule immobilization and enhance stability and activity. The fast electron-transfer kinetics and high conductivity of these materials contribute to improved sensitivity and selectivity. Furthermore, The H-GO/Aptamer composite's large surface area increases the number of immobilized secondary aptamers and enables a more stable structure. This large surface area also facilitates more H loading, leading to signal amplification.

A review of antibody, aptamer, and nanomaterials synergistic systems for an amplified electrochemical signal.

The synergy between biomolecules with inorganic nanomaterials and nanoparticles has been investigated over the past years, primarily to improve biomarker reception, generate signals, and amplify the signals generated. In this paper, several articles on aptamer-based and antibody-based electrochemical biosensors that target antigens were examined. Among the key characteristics identified were the electrochemical platform development, which includes the usage of nanomaterials as electroactive or electrocatalytic labels, crosslinking of the biological agent with inorganic compounds, and electrode coating to provide an electronic source and support efficient electron transfer. A single approach using labeled or unlabeled biological receptors has become advantageous due to its simple architecture and more straightforward application method. However, the dual system approach allows the incorporation of more nanomaterials to boost the signal and add more features to the electrochemical system. The dual system approach uses a capture and reporter probe in a competitive or sandwich detection format. The reporter probe is often labeled by an electroactive or electrocatalytic compound or immobilized in a nanocarrier, resulting in an increase in measured peak current in proportion to the target's concentration. The reported limit of detection and linear range for each platform is presented to assess its efficiency. Generally, the dual system aptasensor showed higher sensitivity, stability, and reproducibility than the immunosensor in comparable settings. The aptasensor showed promising results for the development of point-of-care type applications.

Construction of an Enzymatically Controlled DNA Nanomachine for One-Step Imaging of Telomerase in Living Cells.

Telomerase is a basic reverse transcriptase that maintains the telomere length in cells, and accurate and specific sensing of telomerase in living cells is critical for medical diagnostics and disease therapeutics. Herein, we demonstrate for the first time the construction of an enzymatically controlled DNA nanomachine with endogenous apurinic/apyrimidinic endonuclease 1 (APE1) as a driving force for one-step imaging of telomerase in living cells. The DNA nanomachine is designed by rational engineering of substrate probes and reporter probes embedded with an enzyme-activatable site (i.e., AP site) and their subsequent assembly on a gold nanoparticle (AuNP). Upon recognition and cleavage of the AP site in the substrate probe by APE1, the loop of the substrate probe unfolds, exposing telomeric primer (TP) with the 3'-OH end. Subsequently, the TP is elongated by telomerase at the 3'-OH end to generate a long telomeric product. The resultant telomeric product acts as a swing arm that can hybridize with a reporter probe to initiate the APE1-powered walking reaction, ultimately generating a significantly enhanced fluorescence signal. Notably, endogenous APE1 is used as the driving force of the DNA nanomachine, avoiding the introduction of exogenous auxiliary cofactors into the cellular microenvironment. Owing to the high kinetics and high amplification efficiency of the APE1-powered DNA nanomachine, this strategy enables one-step sensitive sensing of telomerase in vitro and in vivo. It can successfully discriminate telomerase activity between cancer cells and normal cells, screen telomerase inhibitors, and monitor the variations of telomerase activity in living cells, offering a prospective platform for molecular diagnostics and drug discovery.

Screening putative polyester polyurethane degrading enzymes with semi-automated cell-free expression and nitrophenyl probes.

Cell-free expression (CFE) has shown recent utility in prototyping enzymes for discovery efforts. In this work, CFE is demonstrated as an effective tool to screen putative polyester polyurethane degrading enzyme sequences sourced from metagenomic analysis of biofilms prospected on aircraft and vehicles. An automated fluid handler with a controlled temperature block is used to assemble the numerous 30 µL CFE reactions to provide more consistent results over human assembly. In sum, 13 putative hydrolase enzymes from the biofilm organisms as well as a previously verified, polyester-degrading cutinase were expressed using in-house E. coli extract and minimal linear templates. The enzymes were then tested for esterase activity directly in extract using nitrophenyl conjugated substrates, showing highest sensitivity to shorter substrates (4-nitrophenyl hexanoate and 4-nNitrophenyl valerate). This screen identified 10 enzymes with statistically significant activities against these substrates; however, all were lower in measured relative activity, on a CFE volume basis, to the established cutinase control. This approach portends the use of CFE and reporter probes to rapidly prototype, screen and design for synthetic polymer degrading enzymes from environmental consortia. Graphical Abstract.

Amplification-free detection of Mpox virus DNA using Cas12a and multiple crRNAs.

Mpox virus (MPXV) is a zoonotic DNA virus that caused human Mpox, leading to the 2022 global outbreak. MPXV infections can cause a number of clinical syndromes, which increases public health threats. Therefore, it is necessary to develop an effective and reliable method for infection prevention and control of epidemic. Here, a Cas12a-based direct detection assay for MPXV DNA is established without the need for amplification. By targeting the envelope protein gene (B6R) of MPXV, four CRISPR RNAs (crRNAs) are designed. When MPXV DNA is introduced, every Cas12a/crRNA complex can target adifferent site of the same MPXV gene. Concomitantly, the trans-cleavage activity of Cas12a is triggered to cleave the DNA reporter probes, releasing afluorescence signal. Due to the application of multiple crRNAs, the amount of active Cas12a increases. Thus, more DNA reporter probes are cleaved. As a consequence, the detection signals are accumulated, which improves the limit of detection (LOD) and the detection speed. The LOD of the multiple crRNA system can be improved to ~ 0.16pM, which is adecrease ofthe LOD by approximately ~ 27 times compared with the individual crRNA reactions. Furthermore, using multiple crRNAs increases the specificity of the assay. Given the outstanding performance, this assay has great potential for Mpox diagnosis.