CRISPR/Cas9-mediated SERS/colorimetric dual-mode lateral flow platform combined with smartphone for rapid and sensitive detection of Staphylococcus aureus.

Pathogenic bacteria infections pose a significant threat to global public health, making the development of rapid and reliable detection methods urgent. Here, we developed a surface-enhanced Raman scattering (SERS) and colorimetric dual-mode platform, termed smartphone-integrated CRISPR/Cas9-mediated lateral flow strip (SCC-LFS), and applied it to the ultrasensitive detection of Staphylococcus aureus (S. aureus). Strategically, functionalized silver-coated gold nanostar (AuNS@Ag) was prepared and used as the labeling material for LFS assay. In the presence of S. aureus, target gene-induced amplicons can be accurately recognized and unwound by the user-defined CRISPR/Cas9 system, forming intermediate bridges that bind many AuNS@Ag to the test line (T-line) of the strip. As a result, the T-line was colored and a recognizable SERS signal was obtained using a smartphone-integrated portable Raman spectrometer. This design not only maintains the simplicity of visual readout, but also integrates the quantitative capability of SERS, enabling the user to flexibly select the assay mode as needed. With this method, S. aureus down to 1 CFU/mL can be detected by both colorimetric and SERS modes, which is better than most existing methods. By incorporating a rapid extraction procedure, the entire assay can be completed in 45 min. The robustness and practicality of the method were further demonstrated by various real samples, indicating its considerable potential toward reliable screening of S. aureus.

Effects of different dietary patterns during pregnancy on birth outcomes and glucose parameters in women with gestational diabetes mellitus: A systematic review and meta-analysis.

PURPOSE: Dietary interventions are the cornerstone of gestational diabetes mellitus (GDM) treatment. This study aimed to evaluate the effects of dietary patterns during pregnancy on birth outcomes and glucose parameters in women with GDM. METHODS: PubMed, Embase, and The CoChrane Library were searched from the time of database creation to November 30, 2021, along with manual searches. Data analyses were performed using Stata 15.4 software. RESULTS: From 2461 studies, 27 RCTs involving 1923 women were eligible. The pooled results showed that dietary pattern interventions during pregnancy reduced birth weight (WMD: -0.14 kg; 95% CI: -0.24, -0.00), hemoglobin A1 C (HbA1 C) (WMD: -0.19, 95% CI: -0.34, -0.05), and macrosomia incidence (RR 0.65 [95% CI 0.48, 0.88]). Low glycemic index (GI) diet reduced macrosomia incidence (RR 0.31 [95% CI 0.11, 0.93]) and fasting plasma glucose (FPG) levels (WMD: -0.10 mmol/L; 95% CI: -0.14, -0.05); a low carbohydrate (CHO) diet reduced large for gestational age (LGA) incidence (RR 0.33 [95% CI 0.13, 0.82]) and HbA1 C (WMD: -0.32; 95% CI: -0.51, -0.14); dietary approaches to stop hypertension (DASH) diet reduced birth weight (WMD:-0.59 kg; 95% CI: -0.64, -0.55), insulin use (RR 0.31 [95% CI 0.18, 0.56), macrosomia incidence (RR 0.12 [95% CI 0.03, 0.50]), and cesarean sections incidence (RR 0.57 [95% CI 0.40, 0.82]). CONCLUSION: Dietary patterns during pregnancy can improve certain birth outcomes and glycemic parameters. Due to limitations in the quality and number of included studies, the above findings still need to be validated by further randomized controlled trials with high quality and large samples.

CRISPR/Cas9-based point-of-care lateral flow biosensor with improved performance for rapid and robust detection of Mycoplasma pneumonia.

Screening of acute respiratory infections causes serious challenges in urgent point-of-care scenarios where conventional methods are impractical and alternative techniques suffer from low accuracy, poor robustness, and reliance on sophisticated instruments. As an improvement to this paradigm, we report a point-of-care lateral flow biosensor (LFB) based on the recognition property of clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (Cas9) and apply it to the detection of Mycoplasma pneumoniae (M. pneumoniae). The designed biosensor employs CRISPR/Cas9 for secondary recognition after preamplification of target gene using specific primer set, avoiding false positives caused by nontarget factors. The high amplification efficiency and low applicable temperatures of recombinase polymerase amplification brings the detection limit of the biosensor to 3 copies even at a preamplification temperature of 25 °C. Its practical application is further demonstrated with 100% accuracy by testing with 43 M. pneumoniae-infected specimens and 80 uninfected specimens. Additionally, the entire detection, including pretreatment, preamplification, CRISPR/Cas9 recognition, and visual analysis, can be completed in 30 min. Featured with the combination of CRISPR/Cas9 and LFB, the biosensor we developed herein ensures excellent convenience, accuracy, and robustness, which endows promising point-of-care screening potential for infectious pathogens.

Colorimetric aptasensor based on spherical nucleic acid-induced hybridization chain reaction for sensitive detection of exosomes.

Exosomes are one of the most promising biomarkers for tumor diagnosis and prognosis. Therefore, the development of convenient and sensitive exosome sensing strategies is of great significance. Herein, we integrated aptamer-based spherical nucleic acids (SNAs) and hybridization chain reaction (HCR) into a colorimetric aptasensor platform and applied it to the detection of exosomes. In this design, the CD63-specific aptamer pre-immobilized on the microplate was used to capture target exosomes, while the SNAs conjugated with nucleolin-specific aptamer and trigger probe H1 were designed for amplifying signal. In the presence of target exosomes, the SNAs can be attached to the microplate by the bridge effect of exosomes, resulting in the trigger of HCR. This process is accompanied by the formation of abundant G-quadruplex/hemin DNAzyme, enabling the visual quantitative analysis of exosomes. Featured with the dual amplification of SNAs and HCR, the proposed aptasensor achieved a considerable detection limit of 50 particles/µL. The practicability of this method was further verified by testing the different clinical samples. Given the ability of the aptasensor to visually detect exosomes in scenarios lacking instruments and resources, we believe that the aptasensor can be serve as a potential on-site test for liquid biopsy.

Bidirectional motivated bimodal isothermal strand displacement amplifier with a table tennis-like movement for the ultrasensitive fluorescent and colorimetric detection of depression-related microRNA.

An increasing number of studies have highlighted the potential of microRNAs (miRNAs) as physiological indicators of major depressive disorder (MDD). Herein, we developed a bidirectional-motivated bimodal isothermal strand displacement amplifier (BB-ISDA) for the ultrasensitive fluorescent and colorimetric detection of MDD-related miRNA-132. In the BB-ISDA system, a pair of functionalized hairpin probes (HP1 and HP2) with nicking recognition sites are designed to recognize target miRNA. The recognition of target miRNA by HP1 (or HP2) generates copious numbers of nicked triggers by HP1 (or HP2)-based ISDA to recognize HP2 (or HP1) by autonomous strand polymerization, cleavage, and displacement, which in turn induces the subsequent generation of copious numbers of nicked G-quadruplex triggers by HP2 (or HP1)-based ISDA to recognize HP1 (or HP2) along a same line. After many cycles, this bidirectional motivated table-tennis-like movement amplifies the fluorescent signal from HP1 and the colorimetric signal from HP2, simultaneously. The dual-signal output pattern was cross-validated for sensing miRNA-132. Each of the detection modal shows the capability for qualitative and quantitative detection of miRNA-132 with high sensitivity and specificity. The adaptability of the bimodal mechanism was verified via the detection of target miRNA-132 from clinical human blood samples. We envision that this BB-ISDA with dual-signal output for accurate and reliable analysis of miRNA is promising for the molecular diagnosis of human mental diseases.

Fabrication of WPI-EGCG covalent conjugates/gellangum double network emulsion gels by duo-induction of GDL and CaCl2 for colon-controlled Lactobacillus Plantarum delivery.

In this study, we demonstrated the role of whey protein isolate (WPI)/(-)-epigallocatechin-3-gallate (EGCG) covalent conjugates/gellangum double network emulsion gels by duo-induction of glucono-δ-lactone (GDL) and CaCl2 on the digestion viability of Lactobacillus Plantarum powders. We employed a novel one-step continuous cold gelation strategy to product double network emulsion gels. The gellan gum in continuous phase was first induced by CaCl2 to form a gel in a short time. Besides, the GDL was improved covalent cross-linking between WPI-EGCG covalent conjugate Pickering particles or particle-coated oil droplets because of the slowly pH reduction. The first cross-linked gellan gum gels could be more closely integrated into the GDL-induced second gel networks with synergistic role, which promoted the formation of compact and homogeneous networks through hydrophobic interactions and disulfide bonds. Thus, the results provide a promising strategy for probiotic powders encapsulation of double network emulsion gels in oral applications of gel type foods.

CRISPR/Cas9 mediated triple signal amplification platform for high selective and sensitive detection of single base mutations.

Single base mutations detection is crucial for the diagnosis and treatment of cancer. However, the current methods with poor selectivity and sensitivity required large instruments, which are difficult to meet clinical demands. Herein, we develop a CRISPR/Cas9 based visual colorimetric platform to specifically detect all single base mutations. In this strategy, the Recombinase Polymerase Amplification (RPA) was firstly used to amplify the target, and introduced the PAM site in the target DNA sequence by designing the point mutation primer, thus achieving detection for all single base mutations by the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated specific recognition. With the help of CRISPR/Cas9 system, those RPA products can release single strand DNA to hybridize with the padlock probe and trigger rolling circle amplification (RCA). Based on the magnetic separation, HRP-gold nanoparticles complex (hGNPs) and biotin modified probe (Bio-probe) were further used to achieve enhanced visual variations assay by hybridizing with RCA products. Benefiting from the RPA assisted triple signal amplification, this method not only showed enhanced sensitivity with a limit of detection (LOD) as low as 0.2 fM and 0.01% of KRAS-G12D mutation percentage, but the specificity against KRAS-G12D mutation also be synergistically enhanced by combining the CRISPR/Cas9-mediated specific recognition with the specific T4 ligation reaction of RCA system. Furthermore, this system has been successfully used to visually detect genome in serum, suggesting its great potential for point-of-care diagnosis in clinical.

Rapid and simultaneous visual typing of high-risk HPV-16/18 with use of integrated lateral flow strip platform.

A biosensor for rapid and simultaneous visual identification of high-risk human papillomavirus (HPV) genotypes 16 and 18 in clinical samples based on polymerase chain reaction (PCR) integrated lateral flow strip platform was developed. Using an one-step protocol to extract nucleic acid rapidly and the functionalized primer sets specific to HPV-16 and 18 were designed for the simultaneous amplification. In the presence of target HPV genotypes, the corresponding functionalized primer sets will participate in the PCR process and produce numerous duplex functionalized dsDNA amplicons. With the bridge effect of duplex functionalized dsDNA amplicons between gold nanoparticles-fluorescein isothiocyanate antibody conjugates (AuNP-FITC antibody conjugates) and other two antibodies on corresponding test line (T1 or T2), visualized color signals on test lines could be obtained directly visible with a naked eye. Combining the high amplification efficiency of PCR and the visualized sensing of LFS, as low as 700 copies of HPV-16 and 18 DNA were detected simultaneously within 75 min, which can promote application in the resource limited settings. High-risk genotypes of HPV-16 and HPV-18 were easily and simultaneously screened with the amplification-assisted molecular lateral flow strip by on-site observation in the resource-limited settings.

Trigging Isothermal Circular Amplification-Based Tuning of Rigorous Fluorescence Quenching into Complete Restoration on a Multivalent Aptamer Probe Enables Ultrasensitive Detection of Salmonella.

Detection of pathogenic bacteria is of vital significance for combating and preventing infectious diseases. In this work, we developed a multivalent aptamer probe (Multi-VAP)-based trigging isothermal circular amplification (TICA) for rapidly and ultrasensitively detecting Salmonella. In this sensing system, the fluorescence of Multi-VAP was strongly quenched via the dual effect of FRET. Introduction of Salmonella to the system forced the configuration change of Multi-VAP, leading to the occurrence of a TICA responsible for tuning all of the fluorescence-quenched Multi-VAP into a complete restoration state. This prominent feature allows the reasonable combination of a strong background restraint and great target signal amplification into one sensing system, which in turn benefits the improvement of the signal-to-noise ratio to ensure that the system has an ultrahigh sensitivity. Combined with the employment of an aptamer to ensure that it has excellent specificity, the Salmonella can be quantitatively and qualitatively analyzed even from human serum. The total processing merely requires sample addition and incubation. The turnaround time of the complete analysis from "sample-to-result" was within 30 min. With the method to decrease the time to detect and simplify the process to operate, the assay was successfully used as a sensing platform for specific detection of as few as 9 CFU/mL Salmonella.

Rapid and simultaneous visual screening of SARS-CoV-2 and influenza virufses with customized isothermal amplification integrated lateral flow strip.

Due to the similar clinical symptoms of influenza (Flu) and coronavirus disease 2019 (COVID-19), there is a looming infection threat of concurrent Flu viruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this work, we introduce a customized isothermal amplification integrated lateral flow strip (LFS) that is capable performing duplex reverse transcription-recombinase polymerase amplification (RT-RPA) and colorimetric LFS in a sequential manner. With customized amplification primer sets targeted to SARS-CoV-2 (opening reading frame 1a/b and nucleoprotein genes) and Flu viruses (Flu A and Flu B), the platform allows the rapid and simultaneous visual screening of SARS-CoV-2 and Flu viruses (Flu A and Flu B) without cross reactivity, false positives, and false negatives. Moreover, it maximally eases the detection, reduces the detection time (1 h), and improves the assay performance to detect as low as 10 copies of the viral RNA. Its clinical application is powerfully demonstrated with 100% accuracy for evaluating 15 SARS-CoV-2-positive clinical samples, 10 Flu viruses-positive clinical samples, and 5 negative clinical samples, which were pre-confirmed by standard qRT-PCR. We envision this portable device can meet the increasing need of online monitoring the serious infectious diseases that substantially affects health care systems worldwide.

Time-resolved fluorescent lateral flow strip for easy and rapid quality control of edible oil.

Gutter oil is strictly prohibited from being reprocessed back to the catering and food industry. Extensive attention has been paid to rapid screening of gutter oil to guarantee the safety of edible oil. Capsaicin, a special component of condiments, has been adopted as the marker of gutter oil. The time-resolved fluorescent microspheres are utilized for labeling of antibody to capsaicin, which are further applied for the construction of fluorescent lateral-flow-strip (LFS). By simple extraction of capsaicin with ethanol (or liquor) from the edible oil, the capsaicin can be rapid determined with the fluorescent LFS in less than 10 min. As low as 20 ng/mL capsaicin can be visually judged and 2.3 ng/mL is achieved as the detection limit by ImageJ analysis. The illegal gutter oil is also well screened with this time-resolved LFS. This method can be a useful candidate for routine quality monitoring of edible oil and a powerful tool for self-inspection at home.

Periodically programmed building and collapse of DNA networks enables an ultrahigh signal amplification effect for ultrasensitive nucleic acids analysis.

ANALYSIS: of molecular species is needed for applications in diagnosis of infections and genetic diseases. Herein, we demonstrate a target DNA-responsive ultrahigh fluorescence signal-on DNA amplification system via periodically programmed building and collapse of DNA networks. In this system, a pair of oligonucleotides of padlock probe (PP) and palindromic hairpin probe (PHP) are utilized. The presence of target DNA firstly hybridizes with PP, allowing the occurrence of rolling circle amplification (RCA) to produce RCA products with tandem repeats in abundance to bind and unfold numbers of PHPs. The conformational change of PHPs enables the building of DNA networks via the intermolecular palindrome pairing, but then makes the DNA networks collapsed via the palindrome-induced strand displacement polymerization. The displaced RCA products are dynamically reused to undergo periodically programmed multiple rounds of DNA network building and collapse. Depend on the bidirectional DNA assembly and disassembly, a strikingly amplified fluorescence can be collected to ultrasensitive and specific detection of target DNA. The practicability has been demonstrated by evaluating target-spiked human serum, saliva, and urine samples with acceptable recoveries and reproducibility. Therefore, this newly explored method opens a promising avenue for the detection of nucleic acids with low abundance in biochemical analysis and diseases diagnosis.

Simultaneous and accurate visual identification of chicken, duck and pork components with the molecular amplification integrated lateral flow strip.

We propose a visual strategy for simultaneous detection of multiple adulterated components in beef by integration of multiple polymerase chain reaction (mPCR) with the lateral flow strip (LFS). The primer sets for adulterated components are uniquely designed with different nucleic acid tags (NAT), enabling the amplicons with specific wobbled sequences at two opposite ends. The wobbled sequences will precisely hybridize with the pre-immobilized capture probes on T lines (T1, T2 and T3) and C line, contributing to the coloration of LFS. Taking advantages of extraordinary amplification efficiency of PCR and simplicity of LFS, common adulterated components including chicken, duck and pork can be easily detected with LOD as low as 0.01% (wt%), which is comparable to that of quantitative real-time polymerase chain reaction (qPCR) but with more simplified operations and reduced costs. The method can be extended to identification of other components by replacing the functional primer set. This method can be a useful candidate for meat quality control at the resource-limited setups.

Self-assembly of a polythymine embedded activatable molecular beacon for one-step quantification of terminal deoxynucleotidyl transferase activity.

We describe an isothermal, single-reaction, and one-step method for signal-on quantification of terminal deoxynucleotidyl transferase (TdT) activity based on the periodic elongation and assembly of polythymine embedded activatable molecular beacon (PTA-MB) into DNA nanostructures. PTA-MB is easily designed according to the rule of the conventional molecular beacon (MB) but engineered with a polyT composed loop. Upon exposure to the specific target TdT, the MB is first elongated with an adenine-rich (A-rich) long chain so that it can then act as the anchoring substrate to capture many original PTA-MBs along its strand. Their unfolding contributes to preliminary fluorescence emission. Significantly, the assembled PTA-MBs can also be elongated and hybridized with residual free PTA-MBs for the second round of signal amplification. Accordingly, multiple rounds of elongation, assembly, and activation of initial PTA-MBs can lead to the formation of DNA nanostructures and induce a dramatically enhanced fluorescence signal for qualitative and quantitative evaluation of TdT activity. The final assay indicated a limit of detection (LOD) of 0.042 U mL-1 TdT and showed excellent selectivity for TdT versus other common enzymes. Moreover, the practical applicability was validated by direct/absolute quantification of TdT in real biological specimens and accurate monitoring of the activity of TdT pretreated by low/high temperature and heavy metal ions. These findings demonstrated that this functional PTA-MB and its unique assembly behavior is most likely to promote the study of oligonucleotide probe-based DNA assembly, providing a reliable, convenient, and universal platform for precise and point-of-care monitoring of various biomolecules.

Smart engineering of a dual-DNA machine with a high signal-to-noise ratio for one-pot robust and sensitive miRNA signaling.

A target triggered dual-DNA machine, consisting of one rolling circle amplification (RCA)-based circular DNA machine (RCA-CDM) and one cyclical strand displacement amplification (CSDA)-based bidirectional DNA machine (CSDA-BDM), was developed for robust miRNA determination.

Rapid visual sensing and quantitative identification of duck meat in adulterated beef with a lateral flow strip platform.

A novel high-sensitivity authentication method has been demonstrated for the rapid visual detection of adulterated meat based on both the lateral flow strip (LFS) platform and on polymerase chain reaction (PCR). After the rapid extraction of genomic components from meat, the on-site amplification of the target DNA of adulterated duck meat is carried out with the rationally designed functional FITC- and biotin-modified primer set, thereby producing numerous double-stranded DNA (dsDNA) products dually labelled with FITC and biotin. The FITC-labelled terminal end of the products binds to the pre-immobilized FITC antibody on the test line of the strip, and the biotin-labelled terminal end binds to the streptavidin-conjugated gold nanoparticles, resulting in a visible test line on the LFS for the rapid identification of duck meat in adulterated beef. After optimization, an adulteration ratio as low as 0.05% can be easily measured, which is more sensitive than other common adulteration authentication methods and is even comparable to instrumental methods. Moreover, 22 commercial processed meat samples were tested with this new strategy, and 4 adulterated samples were successfully identified by both the classic method and our method. In essence, the present authentication method is simple in design, convenient in operation, and can be easily extended to the identification of other adulteration components just by replacing the modified primers.

A sensitive multiplex PCR protocol for simultaneous detection of chicken, duck, and pork in beef samples.

A rapid and sensitive multiplex PCR assay was developed for simultaneous identification of the adulteration ingredients of chicken, duck and pork in beef. Specific primers for the mitochondrial genes of Cyt b, CO III, ATPase subunit 8/6 and Cyt b of chicken, duck, pork, and beef, respectively, were adopted in the assay. DNA exaction from meat samples was carried out by using magnetic nanoparticles as rapid separation substrates. The multiplex PCR assay showed that the limit of detection was 0.05% for each species. Moreover, the multiplex PCR specifically identified five beef samples adulterated with pork and one beef samples adulterated with chicken among the 35 commercial samples examined, indicating the practicability of this multiplex PCR method for identifying adulterated ingredients of chicken, duck, and pork in commercial beef products.

Lateral Flow Test for Visual Detection of Multiple MicroRNAs.

The authors describe a rapid and low-cost approach for multiplex microRNA(miRNA) assay on lateral flow nucleic acid biosensor (LFNAB). The principle of assay is based on sandwich-type nucleic acid hybridization reactions to produce gold nanoparticle (GNP)-attached complexes (ssDNA-microRNA-ssDNA/GNPs), which are captured and visualized on the test zone of LFNAB. By designing three different test zones on LFNAB, simultaneous detection of microRNA-21, microRNA-155 and microRNA-210 was achieved with an adding-measuring model by using GNP as visual tag. The method was challenged by testing the microRNAs in spiked serum samples with satisfied results. In our perception, the test is a particularly valuable tool for clinical application and biomedical diagnosis, particularly in limited resource settings.

Highly Simple and Sensitive Molecular Amplification-Integrated Fluorescence Anisotropy for Rapid and On-Site Identification of Adulterated Beef.

Fluorescence polarization (FP) signal is a self-referencing fluorescence signal, and it is less dependent on dye concentration and environmental interferences, which makes FP measurement a highly attractive alternative sensing technology to conventional fluorescent detection methods. Here we adopted a strategy for rapid increase of molecular weight to increase the FP signal for the detection of meat adulteration. The molecular weight of fluorescent labeled primers increased rapidly by slight preamplification, and the FP values were varied accordingly. We found a positive correlation between adulteration ratio and the FP signals. Detection limit for adulterated beef can be reached as low as 0.1% (wt %), meeting or better than the most detection requirements. On the basis of this proposed amplification-integrated FP method, both the standard samples and the commercial processed beef samples were successfully authenticated with satisfied results.