Semiarbitrary qPCR for Sensitive Detection of Circulating miRNA via Terminal Deoxynucleotidyl Transferase-Assisted RNA-Primed DNA Polymerization.

Circulating microRNAs (miRNAs) have recently emerged as noninvasive disease biomarkers. Quantitative detection of circulating miRNAs could offer significant information for clinical diagnosis due to its significance in the development of biological processes. In response to the current challenges of circulating miRNA detection, we introduce a sensitive, selective, and versatile circulating miRNA detection strategy using terminal deoxynucleotidyl transferase (TdT)-catalyzed RNA-primed DNA polymerization (TCRDP) coupled with semiarbitrary qPCR (SAPCR). Semiarbitrary qPCR was first developed here to detect long fragment targets with only a short-known sequence or to detect a short fragment target after extension with terminal transferase. Besides, the subsequent results show that TdT has a preference for RNA, particularly for extending RNAs with purine-rich and unstructured ends. Consequently, utilizing this assay, we have successfully applied it to the quantitative analysis of circulating miR-122 in animal models, a sensitive and informative biomarker for drug-induced liver injury, and as low as 200 zmol of the target is detected with desirable specificity and sensitivity, indicating that the TCRDP-SAPCR can offer a promising platform for nucleic acids analysis.

Subchronic feeding study of glyphosate-tolerant maize GG2 with the gr79-epsps and gat genes in Wistar Han RCC rats.

The genetically modified (GM) maize GG2 contains gr79-epsps and gat genes, conferring glyphosate tolerance. The present study aimed to investigate potential effects of maize GG2 in a 90-day subchronic feeding study on Wistar Han RCC rats. Maize grains from GG2 or non-GM maize were incorporated into diets at concentrations of 25% and 50% and administered to Wistar Han RCC rats (n = 10/sex/group) for 90 days. The basal-diet group of rats (n = 10/sex/group) were fed with common commercialized rodent diet. Compared with rats fed with the corresponding non-GM maize and the basal-diet, no biologically relevant differences were observed in rats fed with the maize GG2, according to the results of body weight/gain, feed consumption/utilization, clinical signs, mortality, ophthalmology, clinical pathology (hematology, prothrombin time, urinalysis, serum chemistry), organ weights, and gross and microscopic pathology. Under the conditions of this study, these results indicated that maize GG2 is as safe as the non-GM maize in this 90-day feeding study.

Paper-Based Strip for Ultrasensitive Detection of OSCC-Associated Salivary MicroRNA via CRISPR/Cas12a Coupling with IS-Primer Amplification Reaction.

As the most common malignancy in humans, oral squamous cell carcinoma (OSCC) not only harms the people's health but also undermines their confidence after facial surgery. Early detection and treatment can effectively reduce these damages. The unique collateral trans-cleavage nuclease activity of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system was utilized to realize the detection of nucleic acid with high sensitivity. So, in this work, we designed a point-of-care testing (POCT) platform for the detection of OSCC-associated salivary hsa-miRNA 31-5p (miR-31) via the cascade signal amplification of "invading stacking primer" (IS-primer) amplification reaction (ISAR), CRISPR/Cas12a, and dual-mode paper-based strip (dm-Strip). To amplify the detection signal of trace miR-31, the cascade signal amplification of CRISPR/Cas12a system coupling with ISAR was designed in a one-pot reaction at a constant temperature. The target miR-31 could activate the ISAR to generate numerous DNAs, which would further trigger the trans-cleavage effect of Cas12a to catalyze the nonspecific single-stranded DNA (ssDNA) cleavage. This ssDNA was labeled with digoxin and biotin at the 5' and 3' termini (digoxin/ssDNA/biotin), respectively, which led to generate the naked-eye signal and fluorescent signal of the designed dm-Strip. The whole detection time was 90 min with limit-of-detection (LOD) down to aM level. This ISAR/Cas12a-based dm-Strip (ISAR/Cas12a-dmStrip) allowed for the portable and ultrasensitive detection of miRNA, an important step in early diagnosis of OSCC and biomedical research.

A novel analytical principle using AP site-mediated T7 RNA polymerase transcription regulation for sensing uracil-DNA glycosylase activity.

Uracil DNA glycosylase (UDG) is a highly conserved damage repair glycosylase; the abnormal expression of DNA glycosylase has important research value in many human diseases. Therefore, highly sensitive and specific detection of UDG activity is crucial to biomedical research and clinical diagnosis. In this work, we propose an AP site-mediated T7 RNA polymerase transcription regulation analytical principle for uracil-DNA glycosylase activity analysis. T7 RNA polymerase is highly promoter-specific and only transcribes DNA downstream of the T7 promoter. We have found that modifying the T7 promoter sequence with an AP site can regulate T7 RNA polymerase transcription ability according to different modification sites. In the binding region of the promoter, AP sites greatly inhibit transcription. Moreover, AP sites in the initiation region of the promoter enhance transcription activity. Based on this research, we designed a new transcription substrate template by replacing deoxythymidine (dT) in the T7 RNA polymerase promoter sequence with one tetrahydrofuran abasic site mimic (THF) and one deoxyuridine (dU). The THF site was labeled in the transcription-enhanced region to improve transcription background, and the dU site was labeled in the transcription inhibition region to sense the UDG enzyme. In our strategy, this template can be transcribed into RNAs by T7 RNA polymerase with great multicycle amplifications. When UDG is present, dU is excised to form an AP site. The AP site damages the interaction between T7 RNA polymerase and the T7 promoter, resulting in weak transcription activity. The detection limit of this strategy is as low as 2.5 × 10-4 U mL-1, and it has good selectivity for UDG. In addition, this strategy can also detect UDG activity in complex HeLa cell lysate samples. Therefore, our developed sensor might become a promising technique for UDG activity assay.

High sensitive and multiple detection of acute myocardial infarction biomarkers based on a dual-readout immunochromatography test strip.

Immunochromatography test strip (ICTS) displayed high advantages in screening acute myocardial infarction (AMI) biomarkers. However, the low sensitivity and nonquantitative results seriously limited its clinical application. Herein, we designed a highly sensitive, quantitative and dual-readout ICTS for assaying multiple AMI biomarkers based on magnetic nanoparticles (MNPs) quenching the fluorescence of Cy5, which was labeled on capture antibodies on test (T) lines. The changes of fluorescent intensity caused by MNPs nanoprobes enabled us to sensitively quantify cTnI and CK-MB for early diagnosis of AMI in 15 min with a corresponding detection limit of 0.049 ng/mL and 0.085 ng/mL, respectively. Meanwhile, the aggregations of MNPs on T lines allowed colorimetric readout in 2 min for rapid diagnosis of emergent and severe AMI patients. Furthermore, the detection results of 30 clinical serum samples were coincident with those by electrochemiluminescence immunoassay. So this approach is promising a new avenue for clinical diagnosis and prognosis of AMI.

Isothermal nucleic acid amplification strategy by cyclic enzymatic repairing for highly sensitive microRNA detection.

Technologies enabling highly sensitive and selective detection of microRNAs (miRNAs) are critical for miRNA discovery and clinical theranostics. Here we develop a novel isothermal nucleic acid amplification technology based on cyclic enzymatic repairing and strand-displacement polymerase extension for highly sensitive miRNA detection. The enzymatic repairing amplification (ERA) reaction is performed via replicating DNA template using lesion bases by DNA polymerase and cleaving the DNA replicate at the lesions by repairing enzymes, uracil-DNA glycosylase, and endonuclease IV, to prime a next-round replication. By utilizing the miRNA target as the primer, the ERA reaction is capable of producing a large number of reporter sequences from the DNA template, which can then be coupled to a cyclic signal output reaction mediated by endonuclease IV. The ERA reaction can be configured as a single-step, close-tube, and real-time format, which enables highly sensitive and selective detection of miRNA with excellent resistance to contaminants. The developed technology is demonstrated to give a detection limit of 0.1 fM and show superb specificity in discriminating single-base mismatch. The results reveal that the ERA reaction may provide a new paradigm for efficient nucleic acid amplification and may hold the potential for miRNA expression profiling and related theranostic applications.

Highly sensitive and selective strategy for microRNA detection based on WS2 nanosheet mediated fluorescence quenching and duplex-specific nuclease signal amplification.

MicroRNAs (miRNAs) play vital roles in physiologic and pathologic processes and are significant biomarkers for disease diagnostics and therapeutics. However, rapid, low-cost, sensitive, and selective detection of miRNAs remains a challenge because of their short length, sequence homology, and low abundance. Herein, we report for the first time that WS2 nanosheet can exhibit differential affinity toward short oligonucleotide fragment versus ssDNA probe and act as an efficient quencher for adsorbed fluorescent probes. This finding is utilized to develop a new strategy for simple, sensitive, and selective detection of miRNA by combining WS2 nanosheet based fluorescence quenching with duplex-specific nuclease signal amplification (DSNSA). This assay exhibits highly sensitive and selective with a detection limit of 300 fM and even discriminate single-base difference between the miRNA family members. The result indicates that this simple and cost-effective strategy holds great potential application in biomedical research and clinical diagnostics.

A 90-day feeding study of genetically modified maize LP007-1 in wistar han RCC rats.

LP007-1 is a variety of insect-resistant and herbicide-tolerant maize containing the modified cry1Ab, cry2Ab, vip3Aa and cp4-epsps genes. The food safety assessment of the maize LP007-1 was conducted in Wistar Han RCC rats by a 90-days feeding study. Maize grains from both LP007-1 or its corresponding non-genetically modified control maize AX808 were incorporated into rodent diets at 25% and 50% concentrations by mass and administered to rats (n = 10/sex/group) for 90 days. A commercialized rodent diet was fed to an additional group as the basal-diet group. The diets of all groups were nutritionally balanced. No biologically relevant differences were observed in rats fed with maize LP007-1 compared to rats fed with AX808 and the basal-diet with respect to body weight/gain, food consumption/utilization, clinical signs, mortality, ophthalmology, clinical pathology (hematology, prothrombin time, activation of partial thrombin time, serum chemistry, urinalysis), organ weights, and gross and microscopic pathology. Considering the circumstances of this study, the results provided evidence that LP007-1 maize did not exhibit toxicity in the 90-day feeding study.

MicroRNA-Responsive DNA-Programmed Nanomedicine with Controllability of Cascaded Events for Cancer Therapy Enhancement.

Chemotherapy is a prime tool for cancer clinical therapy. The effectiveness has been improved considerably with the assistance of nanotechnology. However, it still meets the challenge of unsatisfied therapeutic effects caused by multidrug resistance and uncontrollable drug release. For further enhancement of the treatment performance, we develop a kind of microRNA-responsive nanomedicine that uses the biomarker microRNA-21 as a trigger of cascaded killing effects on cancer cells, including chemotherapy and gene silencing. The nanomedicine consists of a gold nanoparticle core and a DNA layer. Strand migrations within the layer can accurately control the events of anticancer drug doxorubicin release and multidrug-resistant-associated protein 1 downregulation, yielding an alleviation of multidrug resistance and enhanced killing on cancer cells. This work demonstrates a microRNA-responsive nanomedicine in combination with chemotherapy and gene silencing, which paves the way to the advancement of DNA-based nanomedicine for cancer theranostics.

Construction of a Novel Biosensor Based on the Self-assembly of Dual-Enzyme Cascade Amplification-Induced Copper Nanoparticles for Ultrasensitive Detection of MicroRNA153.

MicroRNAs (miRNAs) have received extensive attention because of their potential as biomarkers for cancer diagnosis and monitoring, and their effective detection is very significant. Here, a specific, one-pot, rapid, femtomolar sensitive miRNAs detection biosensor was developed based on the target-triggered three-way junction (3-WJ) and terminal deoxynucleotide transferase (TDT)/Nt.BspQI in combination with activated copper nanoparticles (CuNPs) self-assembly. To this end, a 3-WJ hairpin probe and helper probe were designed to selectively identify the target miRNA, so as to form a stable 3-WJ structure that further triggered the double-enzyme cycling to produce poly T to activate the self-assembly of CuNPs. Based on the simplicity of CuNPs generation, the poly T template fluorescence CuNPs can detect the minimum detection limit of 1 fm within 1.75 h. In addition, the applicability of this method in complex samples was demonstrated by analyzing the whole-blood RNA extraction from Parkinson patients, consisting of the results of commercial miRNA kits. The developed strategy performs powerful implications for miRNA detection, which may be beneficial for the effective diagnostic assays and biological research of Parkinson's disease.

High fluorescence quenching probe-based reverse fluorescence enhancement LFTS coupling with IS-primer amplification reaction for the rapid and sensitive Parkinson Disease-associated MicroRNA detection.

Parkinson Disease (PD) is the second-most common neurodegenerative disorder in the population. Recent researches indicated that hsa-microRNA 5010-3p (miR-5010) and hsa-microRNA 331-5p (miR-331) were significantly important for the detection of PD. So, in this work, a kind of high fluorescence quenching probe-based reverse fluorescence enhancement lateral flow test strip (rLFTS) was constructed to realize the synchronous detection of miR-5010 and miR-331. The formation of black hole quencher 2 (BHQ2) coating gold nanoparticles (AuNPs) effectively enhanced the fluorescence quenching property of the probes so as to significantly improve the detection sensitivity. This rLFTS also coupled with "invading stacking primer" (IS-primer) isothermal amplification reaction (ISAR) to accomplish rapid, sensitive, specific, and synchronous detection of PD-associated microRNA (miRNA). The whole detection time was shorter (35 min), and the limit-of-detection (LOD) reached to fM level. For the high accuracy diagnosis of PD, the synchronous determination of miR-5010 and miR-331 was successfully realized on one rLFTS by labeling fluorescent molecules to different T-line. This rLFTS also allowed for miRNA detection in total microRNA extracts from whole blood samples of PD patients, which performed important value in PD diagnosis and biomedical research.

Nonenzyme Cascaded Amplification Biosensor Based on Effective Aggregation Luminescence Caused by Disintegration of Silver Nanoparticles.

Sensitive and portable quantification of biomarkers has particular significance in the monitoring and treatment of clinical diseases. Conventional immunoassays were accustomed to introducing or incorporating enzymes for signal amplification, which commonly suffered from poor stability and inferior tolerance. Herein, we constructed a novel nonenzyme amplification methodology based on fluorogenic Ag+-tetrazolate aggregation coupled with silver corrosion sensitization for biomarker determination. A significant cascade enhancement strategy was achieved by the valid aggregation luminescence caused by the potent disintegration of silver nanoparticles. Furthermore, efficient magnetic separation was also combined and performed for the rapidity and simplicity of operation. As the target, the detection limit of prostate-specific antigen was 15.66 pg/mL in our designed biosensor. Besides, a good linear relationship was obtained. The designed biosensor demonstrated good specificity and was successfully applied to clinical serum sample detection.

Reusable Bioluminescent Sensor for Ultrasensitive MicroRNA Detection Based on a Target-Introducing "Fuel-Loading" Mechanism.

As a kind of important potential biomarkers, the expression level of some microRNAs (miRNAs) is closely related to cancer development and progression. Herein, a reusable ultra-sensitive "fuel-loadings" bioluminescent sensor was constructed to detect the trace miRNA based on the cascading signal amplification, which combined the target-introducing "fuel-loading" mechanism and cyclic bioluminescence assay. In this sensor, magnetic beads labeled with hairpin DNA probes (hDNA) could specifically hybridize with the target miRNA and isolate these targets from samples. Then, the target-introducing "fuel loading" mechanism worked because the poly(A) polymerase can catalyze the template-independent sequential addition of adenosine monophosphate (AMP) to the 3' ends of the miRNA targets to produce long poly(A) tails. The long poly(A) tails provided lots of 5'AMPs (cleaved by Exonuclease T), which further as fuels were converted into adenosine-triphosphate (ATP) to generate an enhanced bioluminescent signal by cyclic AMP pyrophosphorylation-ATP dephosphorylation. The "fuel-loadings" bioluminescent sensor realized a high sensitivity with a limit-of-detection of about 22.6 aM for miRNA 21. Moreover, this "fuel-loadings" bioluminescent sensor not only achieved regenerable and reusable measurement in the same microwell to decrease the analysis costs, but also could directly detect miRNA 21 in the serum without complicated extraction procedures. It showed excellent coherence with quantitative reverse transcription polymerase chain reaction for miRNA 21 detection of cancer patients' samples, indicating clinical translation potential for miRNA detection.

A novel template repairing-PCR (TR-PCR) reaction platform for microRNA detection using translesional synthesis on DNA templates containing abasic sites.

A novel template repairing-PCR technology based on miRNA-primed bypass synthesis at the abasic sites on the PCR template is developed as a sensitive and selective platform for miRNA detection. The assay is expected to show great promise for reverse transcription-free RNA PCR amplification and target-based coding analysis.

Enzyme-free colorimetric detection of MicroRNA-21 using metal chelator as label for signal generation and amplification.

MicroRNAs (miRNAs) were reported to be potential tumor markers for early diagnosis of cancer. Due to its short sequence, low expression level and high susceptibility to degradation, the stable and sensitive detection method of miRNAs is arduous to establish. In this work, we designed a metal chelator (ethylenediamine tetraacetic acid disodium salt, EDTA•2Na) labeled oligonucleotides as the plasmonic signal supraregulator probe to control the generation of gold nanoparticles (AuNPs). Based on another complementary oligonucleotides of target miRNA labeling SiO2 microparticles (SiO2MPs) as the detecting platform, EDTA•2Na labeled oligonucleotide probes were immobilized on the SiO2 platform through the sandwich structure in the presence of target miRNA. The sandwich chelating device could further chelate Au3+ to regulate the generation of AuNPs, resulting in colorimetric signal to qualitatively and quantitatively detect the concentration of microRNA-21 (miR-21). The results indicate that the proposed metal chelator -labeled signal amplification method has outstanding sensitivity (LOD = 8.9 fM) and excellent stability, which will be benefit for the early accurate diagnosis of miRNAs.

A review of fluorescent signal-based lateral flow immunochromatographic strips.

Fluorescent signal-based lateral flow immunochromatographic strips (FLFICS) have received great expectations since they effectively improve detection sensitivity with quantitative analysis, and still retain the advantages of simplicity, rapidness, and portability of a common lateral flow immunochromatographic strip (LFICS). Diverse fluorescent reporters have promoted development of FLFICS, such as fluorescent dyes, quantum dots (QDs), an up-converting phosphor (UCP), lanthanide labels, and other fluorescence nanoparticles. In this work, we discuss the different fluorescent reporters applied with LFICS and their unique properties as well as signal amplification strategies helping to enhance detection performance. Benefitting from these sensitive and accurate fluorescent labels, FLFICS commendably satisfies requirements for detecting different disease biomarkers in medical diagnosis, strict supervision of food safety, and water pollution. This work also gives a short introduction to trends in the development of future FLFICS technology.

Single-base mismatch discrimination by T7 exonuclease with target cyclic amplification detection.

T7 exonuclease is reported for the first time to have high specificity in discriminating single-base mismatch and utilized for developing a target cyclic amplification biosensor strategy for sensitive SNP detection based on graphene oxide quenching of uncleaved probes.

A sensitive, homogeneous fluorescence assay for detection of thymine DNA glycosylase activity based on exonuclease-mediated amplification.

A novel homogeneous fluorescence assay strategy for highly sensitive detection of thymine DNA glycosylase (TDG) enzyme activity based on the exonuclease-mediated signal amplification reaction was reported.

Target-mediated consecutive endonuclease reactions for specific and sensitive homogeneous fluorescence assay of O6-methylguanine-DNA methyltransferase.

O(6)-Methylguanine-DNA methyltransferase (MGMT) is one of the most important DNA-repair enzymes. Herein, a simple, sensitive and selective homogeneous fluorescence assay strategy is developed for the detection of MGMT on the basis of target-mediated two consecutive endonuclease reactions. The activity assay of MGMT is firstly accomplished using a hairpin-structured DNA substrate to offer a specific recognition site on the substrate DNA for restriction endonuclease PvuII, and thus to initiate the first endonuclease reaction. The product which activates the second endonuclease reaction allows an efficient amplification approach to create an abundance of fluorescence signal reporters. The first endonuclease reaction offers the method high specificity and the second one furnishes the assay improved sensitivity. The results reveal that the MGMT assay strategy shows dynamic responses in the concentration range from 1 to 120 ng mL(-1) with a detection limit of 0.5 ng mL(-1). By simply altering the alkylated bases, this strategy can also be extended for the detection of other alkyltransferases. Therefore, the developed strategy might provide an intrinsically convenient, sensitive and specific platform for alkyltransferase activate assay and related biochemical studies due to its label-free, homogeneous, and fluorescence-based detection format.