Simultaneous Detection of Micro-RNAs by a Disposable Biosensor via the Click Chemistry Connection Strategy.

Simultaneous detection of multiple breast cancer-associated miRNAs significantly raises the accuracy and reliability of early diagnosis. In this work, disposable carbon fiber paper serves as the biosensing interface, linking DNA probes via click chemistry to efficiently capture targets and signals efficiently. DNA probes have multiple recognition domains that trigger a cascade reaction through the helper probes and targets, resulting in two signals output. The signals are centrally encapsulated in the pore of the MIL-88(Fe)-NH2. The signal carriers are directed by signal probes to the recognition domains that correspond to the DNA probes. The biosensor is selective and stable, and it can quantify miRNA-21 and miRNA-155 simultaneously with detection limits of 0.64 and 0.54 fmol/L, respectively. Furthermore, it demonstrates satisfactory performance in tests conducted with normal human serum and cell lysate. Overall, this method makes a satisfactory exploration to realize an inexpensive and sensitive biosensor for multiple biomarkers.

A Clinically Feasible Diagnostic Electrochemical Micronano Motors Biosensor Built on Miniature Swimmer for Multiplex Detection and Grading of Breast Cancer Biomarkers.

Estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER2), and Ki67 are four crucial biomarkers used in the clinical diagnosis of breast cancer. Accurate detection of these biomarkers is essential for an effective diagnosis and treatment. MOF-based micronano motors (MOFtors) are promising for various applications, including environmental remediation, targeted nanosurgery, and biomarker detection. This paper presents a clinically feasible diagnostic electrochemical micronano motor biosensor, built on a miniature swimmer, for the multiplex detection and grading of breast cancer biomarkers. We designed a biosensor, named MOFtor-MSEM, incorporating aptamers and antibodies functionalized on SiO2@Co-Fe-MOF, which acts as a miniature swimmer in solution. The SiO2@Co-Fe-MOF serves as the body, while complementary double-chain-linked antibodies function as paddles. In a homogeneous solution, when a positive voltage is applied to the working electrode, the electrostatic interaction between the neutral SiO2@Co-Fe-MOF and the negatively charged complementary double-linked antibody causes the antibody to move toward the electrode and then regress due to water resistance. This back-and-forth motion propels the miniature swimmer, enabling it to move the target analyte through the solution. The sensor features an automatic "sample-amplifying signal-output" process, achieving simultaneous signal amplification and output of four electrochemical signals on a single nanomaterial, a significant challenge in electrochemical sensing. The biosensor boasts a short detection time of 40 min, compared to approximately 1 week for current clinical tissue testing. Additionally, the bioplatform selectively detects HER2, ER, Ki67, and PR in the range of 0-1500 pg/mL, with detection limits of 0.01420, 0.03201, 0.01430, and 0.01229 pg/mL, respectively.

A Novel CRISPR/Cas12a-Mediated Ratiometric Dual-Signal Electrochemical Biosensor for Ultrasensitive and Reliable Detection of Circulating Tumor Deoxyribonucleic Acid.

Circulating tumor DNA (ctDNA) holds great promise as a noninvasive biomarker for cancer diagnosis, treatment, and prognosis. However, the accurate and specific quantification of low-abundance ctDNA in serum remains a significant challenge. This study introduced, for the first time, a novel exponential amplification reaction (EXPAR)-assisted CRISPR/Cas12a-mediated ratiometric dual-signal electrochemical biosensor for ultrasensitive and reliable detection of ctDNA. To implement the dual-signal strategy, a signal unit (ssDNA-MB@Fc/UiO-66-NH2) was prepared, consisting of methylene blue-modified ssDNA as the biogate to encapsulate ferrocene signal molecules within UiO-66-NH2 nanocarriers. The presence of target ctDNA KRAS triggered EXPAR amplification, generating numerous activators for Cas12a activation, resulting in the cleavage of ssDNA-P fully complementary to the ssDNA-MB biogate. Due to the inability to form a rigid structure dsDNA (ssDNA-MB/ssDNA-P), the separation of ssDNA-MB biogate from the UiO-66-NH2 surface was hindered by electrostatic interactions. Consequently, the supernatant collected after centrifugation exhibited either no or only a weak presence of Fc and MB signal molecules. Conversely, in the absence of the target ctDNA, the ssDNA-MB biogate was open, leading to the leakage of Fc signal molecules. This clever ratiometric strategy with Cas12a as the "connector", reflecting the concentration of ctDNA KRAS based on the ratio of the current intensities of the two electroactive signal molecules, enhanced detection sensitivity by at least 60-300 times compared to single-signal strategies. Moreover, this strategy demonstrated satisfactory performance in ctDNA detection in complex human serum, highlighting its potential for cancer diagnosis.

Label-Free and DNAzyme-Mediated Biosensor with a High Signal-to-Noise Ratio for a Lumpy Skin Disease Virus Assay.

Lumpy skin disease virus (LSDV) is a severe and highly contagious form of cowpox. As LSDV continues to mutate and there is no vaccine and treatment in nonendemic countries, early detection of LSDV becomes an important basis for epidemic prevention and control, especially for detection of conserved sequences. A new label-free and sensitive fluorescence method was developed based on a light-up RNA aptamer for detecting LSDV. The method integrated recombinase polymerase amplification (RPA), CRISPR/Cas12a, 10-23 DNAzyme, and Baby Spinach RNA aptamer for triple cascade signal amplification. Based on highly sensitive and specific RPA and CRISPR/Cas12a, DNAzyme achieved a third signal amplification. Additionally, the Baby Spinach RNA aptamer had stronger fluorescence signals and higher quantum yields. The label-free method had ultrahigh sensitivity with the actual detection limit as 1.29 copies·µL-1. The method was 100-fold more sensitive compared to RPA with Cas12a. Moreover, it had no cross-reactivity with viruses belonging to the Capripoxvirus, such as sheep pox virus and goat pox virus with genetic homology as 97%. Furthermore, the method displayed 100% accuracy in 50 actual samples. Therefore, the method based on RPA, Cas12a, and 10-23 DNAzyme had advantages in LSDV detection and provided a new solution for LSD prevention and control.

Fe Single-Atom Carbon Dots Nanozyme Collaborated with Nucleic Acid Exonuclease III-Driven DNA Walker Cascade Amplification Strategy for Circulating Tumor DNA Detection.

Circulating tumor DNA (ctDNA), as a next-generation tumor marker, enables early screening and monitoring of cancer through noninvasive testing. Exploring the development of new methods for ctDNA detection is an intriguing study. In this work, a unique electrochemical biosensor for the ctDNA detector was constructed in the first utilizing Fe single-atom nanozymes-carbon dots (SA Fe-CDs) as a signaling carrier in collaboration with a DNA walker cascade amplification strategy triggered by nucleic acid exonuclease III (Exo III). The electrochemical active surface area of AuNPs/rGO modified onto a glassy carbon electrode (AuNPs/rGO/GCE) was about 1.43 times that of a bare electrode (bare GCE), with good electrical conductivity alongside a high heterogeneous electron transfer rate (5.81 × 10-3 cm s-1), that is, as well as the ability to load more molecules. Sequentially, the DNA walker cascade amplification strategy driven by Exo III effectively converted the target ctDNA into an amplified biosignal, ensuring the sensitivity and specificity of ctDNA. Ultimately, the electrochemical signal was further amplified by introducing SA Fe-CDs nanozymes, which could serve as catalysts for 3,3',5,5'-tetramethylbenzidine (TMB) oxidation with facile responding (Vmax = 0.854 × 10-6 M s-1) and robust annexation (Km = 0.0069 mM). The integration of the triple signal amplification approach achieved detection limits as low as 1.26 aM (S/N = 3) for a linearity spanning from 5 aM to 50 nM. In this regard, our proposal for a biosensor with exceptional assay properties in complicated serum environments had great potential for early and timely diagnosis of cancer.

CRISPR/Cas12a-coupled multiplexed strand displacement amplification for miRNA155 one-tube detection: via a dual-cavity PCR tube.

A CRISPR/Cas12a-coupled multiplexed strand displacement amplification (CMSDA) for the detection of miR155 has been developed. Non-specific amplification was avoided by designing a single-stranded DNA template with a hairpin structure. The detection target miR155 was used as a primer to initiate a multiple-strand displacement reaction to produce abundant ssDNA. ssDNA was recognized by the Cas12a/CrRNA binary complex, activating the trans-cleaving activity of Cas12a. The multiple-strand displacement reaction is more efficiently detected compared with a single-strand displacement reaction. The detection range is from 250 pM to 1 nM, and the limit of the detection is 6.5 pM. The proposed method showed a good applicability in complex serum environments, indicating that the method has a broad prospect for disease detection and clinical application. In addition, we designed a dual-cavity PCR tube, which realized one-tube detection of miRNA155 and avoided open-cap contamination.

Histidine modified Fe3O4 nanoparticles improving the ethanol yield and tolerance of Saccharomyces cerevisiae.

Saccharomyces cerevisiae, the primary microorganism involved in ethanol production, is hindered by the accumulation of ethanol, leading to reduced ethanol production. In this study, we employed histidine-modified Fe3O4 nanoparticles (His-Fe3O4) for the first time, to the best of our knowledge, as a method to enhance ethanol yield during the S. cerevisiae fermentation process. The results demonstrated that exposing S. cerevisiae cells to Fe3O4 nanoparticles (Fe3O4 NPs) led to increased cell proliferation and glucose consumption. Moreover, the introduction of His-Fe3O4 significantly boosted ethanol content by 17.3% (p < 0.05) during fermentation. Subsequent findings indicated that the increase in ethanol content was associated with enhanced ethanol tolerance and improved electron transport efficiency. This study provided evidence for the positive effects of His-Fe3O4 on S. cerevisiae cells and proposed a straightforward approach to enhance ethanol production in S. cerevisiae fermentation. The mediation of improved ethanol tolerance offers significant potential in the fermentation and bioenergy sectors.

CRISPR/Cas12a-Powered EC/FL Dual-Mode Controlled-Release Homogeneous Biosensor for Ultrasensitive and Cross-Validated Detection of Messenger Ribonucleic Acid.

Accurate detection of cancer-associated mRNAs is beneficial to early diagnosis and potential treatment of cancer. Herein, for the first time, we developed a novel CRISPR/Cas12a-powered electrochemical/fluorescent (EC/FL) dual-mode controlled-release homogeneous biosensor for mRNA detection. A functionalized ssDNA P2-capped Fe3O4-NH2 loaded with methylene blue (P2@MB-Fe3O4-NH2) was synthesized as the signal probe, while survivin mRNA was chosen as the target RNA. In the presence of the target mRNA, the nicking endonuclease-mediated rolling circle amplification (NEM-RCA) was triggered to produce significant amounts of ssDNA, activating the collateral activity of Cas12a toward the surrounding single-stranded DNA. Thus, the ssDNA P1 completely complementary to ssDNA P2 was cleaved, resulting in that the ssDNA P2 bio-gate on Fe3O4-NH2 could not be opened due to electrostatic interactions. As a result, there was no or only a little MB in the supernatant after magnetic separation, and the measured EC/FL signal was exceedingly weak. On the contrary, the ssDNA P2 bio-gate was opened, enabling MB to be released into the supernatant, and generating an obvious EC/FL signal. Benefiting from the accuracy of EC/FL dual-mode cross-verification, high amplification efficiency, high specificity of NEM-RCA and CRISPR/Cas12a, and high loading of mesoporous Fe3O4-NH2 on signal molecules, the strategy shows aM-level sensitivity and single-base mismatch specificity. More importantly, the practical applicability of this dual-mode strategy was confirmed by mRNA quantification in complex serum environments and tumor cell lysates, providing a new way for developing a powerful disease diagnosis tool.

The End of the Gray Zone: One-Tube Nested Recombinase Polymerase Amplification with Ultrahigh Signal-to-Noise Ratio for Precisely Detecting and Surveilling Viruses.

The samples were difficult to accurately determine positive or negative between 35 and 40 cycles by real-time quantitative PCR (qPCR) as the standard method. Here, we developed one-tube nested recombinase polymerase amplification (ONRPA) technology with CRISPR/Cas12a to overcome this difficulty. ONRPA broke the amplification plateau to substantially enhance the signals, which considerably improved the sensitivity and eliminated the problem of gray area. Using two pairs of primers one after another, it improved precision by lowering the probability of magnifying several target zones, which was completely free of contamination by nonspecific amplification. This was important in nucleic acid testing. Finally, by the CRISPR/Cas12a system as a terminal output, the approach achieved a high signal output as few as 2.169 copies·µL-1 in 32 min. ONRPA was 100-fold more sensitive than conventional RPA and 1000-fold compared to qPCR. ONRPA coupled with CRISPR/Cas12a will be an important and new promoter of RPA in clinical applications.

Microswimmer-Based Ultrasensitive Dual-Signal Ratiometric Electrochemical Homogeneous Aptasensor with Functionalized Co-UiO-66 for the Single-Step Detection of HER2 in Whole Blood.

The ability to efficiently detect trace disease biomarkers in whole blood remains an enormous challenge. Researchers have paid more attention to the homogeneous electrochemical ratio biosensor due to its self-calibration capability and improved detection accuracy. However, proportional homogeneous electrochemical sensing is difficult to achieve and typically requires functional modification of the electrode or the preparation of complex materials. Herein, a dual-signal ratiometric aptamer homogeneous electrochemical microswimming detection device with active capture capability and one-step detection of human epidermal growth factor receptor-2 (HER2) is proposed. The homogeneous electrochemical biosensor is fabricated based on a functionalized nanocomposite double-stranded DNA({single-stranded DNA-ferrocene (Fc)-aptamer})@Co-UiO-66 with catalase properties and adsorptive properties to electroactive toluidine blue (TB) molecules. Encapsulation of Co-UiO-66 material with dsDNA (ssDNA-Fc-Apt) containing HER2 aptamer as a gate switch inhibited its ability to adsorb TB molecules. This functionalized Co-UiO-66 material can catalyze hydrogen peroxide. Using hydrogen peroxide as a fuel, it breaks down to release oxygen bubbles, creating a propulsion force that drives dsDNA(ssDNA-Fc-Apt)@Co-UiO-66 target HER2 through whole blood. When the surface dsDNA (ssDNA-Fc-Apt)@Co-UiO-66 recognizes HER2, a strand displacement reaction occurs, and the ssDNA-Fc is released into solution. The HER2 aptamer is coiled because it targets HER2, and the ability to adsorb TB molecules is restored due to the exposed surface of Co-UiO-66. A certain negative voltage is applied to the ITO electrode, and due to the electrostatic attraction, the TB molecules and ssDNA-Fc are adsorbed and enriched on the surface of the electrode by electrostatic attraction, which produces two strong and oppositely changing electrochemical signals, and the electrochemical signals depend on the HER2 concentration. It can sensitively detect HER2 biomarkers in only 40 min with the detection range of 0.0001-10 ng/mL and detection limits as low as 10 fg/mL. The electrochemical microswimmer for the detection of trace disease biomarkers involves a one-step process of capture, signal change, and detection.

Microswimmer-Assisted Dual-Signal Sensor for Multiple Targets in Whole Blood.

Accurate detection of biomarkers in whole blood is an important aspect of diagnostic testing but remains a challenge due to various interferences. However, using a self-calibrating two-signal strategy offers a solution that can overcome interference caused by experimental and environmental factors. Here, we proposed a novel microswimmer {methylene blue (MB)@ZIF-90@aptamer-HER2/3,3',5,5'-tetramethylbenzidine (TMB)@ZIF-90@aptamer-ER}-dual-signal (electrochemical and fluorescence) homogeneous sensor based on functionalized ZIF nanomaterials for one-step simultaneous detection of human epidermal growth factor receptor-2 (HER2) and estrogen receptor (ER) in whole blood. The proposed one-step ZIF-90 synthesis encapsulates TMB and MB with dual-signal properties. HER2 and ER aptamers adsorbed on MB@ZIF-90/TMB@ZIF-90 function as the gate switches. The microswimmer targets the HER2 and ER with adenosine triphosphate (ATP)-driven motion. When targets are present, aptamers dissociate and reduce the microswimmer's surface negative charge. The microswimmer undergoes attack and decomposition by swimming ATP due to the strong coordination force between ATP and Zn2+, leading to the release of MB and TMB. The negative charges on the surface of indium tin oxide enrich MB and TMB with positive charges, thereby increasing the intensities of electrochemical and fluorescence signals. The detection process was completed within 40 min, and the detection limits for ER and HER2 were 8.1 and 5.7 fg/mL respectively, with a linear range of 0.25-20 pg/mL.

A label-free fluorescent sensor for rapid and sensitive detection of ctDNA based on fluorescent PDA nanoparticles.

Technological advances in the detection of circulating tumor DNA (ctDNA) have made new options available for diagnosis, classification, biological studies, and treatment selection. However, effective and practical methods for analyzing this emerging class of biomarkers are still lacking. In this work, a fluorescent biosensor was designed for the label-free detection of ctDNA (EGFR 19 del for non-small cell lung cancer, NSCLC). The biosensor was based on the fact that MnO2 nanosheets (MnO2 NSs) have stronger affinity towards single-stranded DNA (ssDNA), as compared with double-stranded DNA (dsDNA). As a high-performance nanoenzyme, MnO2 NSs could oxidize dopamine (DA) into fluorescent polydopamine nanoparticles (FL-PDA NPs), which could be used as a fluorescence signal. The probe ssDNA could be adsorbed on the surface of MnO2 NSs through π-π stacking, and the active site would be masked, causing a lower fluorescence. After the targets were recognized by probe ssDNA to form dsDNA, its affinity for MnO2 NSs decreased and the active site recovered, causing a restored fluorescence. It was verified that Mn ions, •OH radicals and electron transfer were the important factors in the catalytic oxidation of DA. Under the optimal experimental conditions, this biosensor exhibited a detection limit of 380 pM and a linear range of 25-125 nM, providing reliable readout in a short time (45 min). This sensor exhibited outstanding specificity, stability and reproducibility. In addition, this sensor was applied to the detection of ctDNA in serum samples and cell lysates. It is demonstrated that FL-PDA NPs can be used as a fluorescence signal for easy, rapid and label-free detection of ctDNA without any other amplification strategies, and the proposed strategy has great potential for biomarker detection in the field of liquid biopsy.

A PVDF-based colorimetric sensor array for noninvasive detection of multiple disease-related volatile organic compounds.

Detection of human-generated volatile organic compounds (VOCs) is a new pathway for assessing health. Herein, a polyvinylidene fluoride (PVDF)-based colorimetric sensor array was designed for detecting disease-related VOCs (DVOCs) within 15 min, using a complex of Cu metal-organic framework, graphene aerogel, and dyes as response materials. Fingermaps derived from 28 DVOCs were obtained for further data processing. Pattern recognition was successfully employed in the correct discrimination of 28 DVOCs in low (10 µM), medium (100 µM), and high (300 µM) concentrations. Importantly, the sensor array also presented excellent discrimination ability and application potential when detecting VOCs produced by human cancer and normal cells. In general, VOC acquisition is noninvasive and harmless, and the PVDF-based sensor arrays are simple and visual. Such advantages expand their further application potential.

An ultrasensitive aptasensor of SARS-CoV-2 N protein based on ion current rectification with nanopipettes.

Since the outbreak of COVID-19 in the world, it has spread rapidly all over the world. Rapid and effective detection methods have been a focus of research. The SARS-CoV-2 N protein (NP) detection methods currently in use focus on specific recognition of antibodies, but the reagents are expensive and difficult to be produced. Here, aptamer-functionalized nanopipettes utilize the unique ion current rectification (ICR) of nanopipette to achieve rapid and highly sensitive detection of trace NP, and can significantly reduce the cost of NP detection. In the presence of NP, the surface charge at the tip of the nanopipette changes, which affects ion transport and changes the degree of rectification. Quantitative detection of NP is achieved through quantitative analysis. Relying on the high sensitivity of nanopipettes to charge fluctuations, this sensor platform achieves excellent sensing performance. The sensor platform exhibited a dynamic working range from 102-106 pg/mL with a detection limit of 73.204 pg/mL, which showed great potential as a tool for rapidly detecting SARS-CoV-2. As parallel and serial testing are widely used in the clinic to avoid missed diagnosis or misdiagnosis, we hope this platform can play a role in controlling the spread and prevention of COVID-19.

Ratio fluorescence sensor based on CD/Cu-MOFs for detection of Hg2.

Trace detection of toxic heavy metals is a very important and difficult problem in several areas: convenience, sensitivity, and reliability. Herein, we develop an innovative fluorescence resonance energy transfer (FRET)-based ratio fluorescence sensor for the detection of heavy metal mercury ion (H g 2+). The sensing platform is composed of coumarin derivatives (CDs) and a copper metal-organic framework (Cu-MOF) named CD/Cu-MOF. The constructed CD/Cu-MOFs ratio fluorescence sensor exhibits dual emission peaks at 430 and 505 nm under the single excitation wavelength of 330 nm. With the addition of H g 2+, the fluorescence intensity of the system at 430 nm gradually increased, and the fluorescence intensity at 505 nm remained stable, resulting in a change in the fluorescence ratio. There is a good logarithmic relationship between the H g 2+ concentration in the range from 2×10-8 to 0.001 nM and the ratio of the fluorescence emission intensity of the system (F 430/F 505) (R 2=0.9901), and its calculated detection limit is 3.76×10-9 n M. In addition, the CD/Cu-MOFs ratio fluorescence sensor has achieved a good recovery rate of standard addition in the actual food sample recovery experiment, which provides an effective method for the detection of H g 2+ in food samples.

Electrochemical detection of the p53 gene using exponential amplification reaction (EXPAR) and CRISPR/Cas12a reactions.

An improved electrochemical sensor has been developed for sensitive detection of the p53 gene based on exponential amplification reaction (EXPAR) and CRISPR/Cas12a. Restriction endonuclease BstNI is introduced to specifically identify and cleave the p53 gene, generating primers to trigger the EXPAR cascade amplification. A large number of amplified products are then obtained to enable the lateral cleavage activity of CRISPR/Cas12a. For electrochemical detection, the amplified product activates Cas12a to digest the designed block probe, which allows the signal probe to be captured by the reduced graphene oxide-modified electrode (GCE/RGO), resulting in an enhanced electrochemical signal. Notably, the signal probe is labeled with large amounts of methylene blue (MB). Compared with traditional endpoint decoration, the special signal probe effectively amplifies the electrochemical signals by a factor of about 15. Experimental results show that the electrochemical sensor exhibits wide ranges from 500 aM to 10 pM and 10 pM to 1 nM, as well as a relatively low limit detection of 0.39 fM, which is about an order of magnitude lower than that of fluorescence detection. Moreover, the proposed sensor shows reliable application capability in real human serum, indicating that this work has great prospects for the construction of a CRISPR-based ultra-sensitive detection platform.

Catalytic Hairpin Assembly-Driven Ratiometric Dual-Signal Electrochemical Biosensor for Ultrasensitive Detection of MicroRNA Based on the Ratios of Fe-MOFs and MB-GA-UiO-66-NH2.

In this work, a novel ratio electrochemical biosensing platform based on catalytic hairpin assembly target recovery to trigger dual-signal output was developed for ultrasensitive detection of microRNA (miRNA). To achieve the ratiometric dual-signal strategy, methylene blue (MB), an electrochemical indicator, was ingeniously loaded into the pores of graphene aerogel (GA) and metal-organic framework (MOF) composites with high porosity and large specific surface area, and another electrochemical indicator Fe-MOFs with distinct separation of redox potential was selected as a signal probe. Concretely, with the presence of the target miRNA, the CHA process was initiated and the signal probe was introduced to the electrode surface, producing abundant double-stranded H1-H2@Fe-MOFs-NH2. Then, the measurement and analysis of the prepared ratiometric electrochemical biosensor by differential pulse voltammetry (DPV) showed that the introduction of the target miRNA led to an increase in the oxidation peak signal of Fe-MOFs (+0.8 V) and a decrease in the oxidation peak signal of MB (-0.23 V). Therefore, the peak current ratio of IFe-MOFs/IMB could be employed to accurately reflect the actual concentration of miRNA. Under optimal conditions, the detection limit of the proposed biosensor was down to 50 aM. It was worth noting that the proposed biosensor exhibited excellent detection performance in a complex serum environment and tumor cell lysates, showing great potential in biosensing and clinical diagnosis.

Rapid and Ultrasensitive Approach for the Simultaneous Detection of Multilocus Mutations to Distinguish Rifampicin-Resistant Mycobacterium tuberculosis.

The untested empirical medications exacerbated the development of multidrug-resistant Mycobacterium tuberculosis (MDR-TB). Here, we develop a rapid and specific method based on loop-mediated isothermal amplification and duplex-specific nuclease for distinguishing rifampicin-resistant M. tuberculosis. Three probes were designed for the codons 516, 526, and 531 on the RNA polymerase ß-subunit (rpoB) gene. These three sites accounted for more than 90% of the total mutations of the ropB gene in the rifampicin-resistant strain. The approach can perform simultaneous and sensitive detection of three mutant sites with the actual detection limit as 10 aM of DNA and 62.5 cfu·mL-1 of bacteria in 67 min under isothermal conditions. Moreover, the positive mode of the approach for MDR-TB can not only deal with the randomness and diversity of mutations but also provide an easier way for medical staff to read the results. Therefore, it is a particularly valuable method to handle major and urgent MDR-TB diagnostics.

Improvement of Lignocellulolytic Enzyme Production Mediated by Calcium Signaling in Bacillus subtilis Z2 under Graphene Oxide Stress.

An increase in exoenzyme production can be enhanced by environmental stresses such as graphene oxide (GO) stress, but the link between the two events is still unclear. In this work, the effect of GO as an environmental stress factor on exoenzyme (lignocellulolytic enzyme, amylase, peptidase, and protease) biosynthesis was investigated in Bacillus subtilis Z2, and a plausible mechanism by which cytosolic Ca2+ regulates lignocellulolytic enzyme production in B. subtilis Z2 subjected to GO stress was proposed. The filter paper-hydrolyzing (FPase [representing total cellulase]), carboxymethylcellulase (CMCase [representing endoglucanase]), and ß-glucosidase activities and extracellular protein concentration of the wild-type strain under 10 µg/mL GO stress were 1.37-, 1.64-, 1.24-, and 1.16-fold those of the control (without GO stress), respectively. Correspondingly, the transcription levels of lignocellulolytic enzyme genes, cytosolic Ca2+ level, and biomass concentration of B. subtilis were all increased. With lignocellulolytic enzyme from B. subtilis used to hydrolyze alkali-pretreated rice straw, the released reducing sugar concentration reached 265.53 mg/g, and the removal rates of cellulose, hemicellulose, and lignin were 52.4%, 30.1%, and 7.5%, respectively. Furthermore, transcriptome data revealed that intracellular Ca2+ homeostasis played a key role in regulating the levels of gene transcription related to the synthesis of lignocellulolytic enzymes and exoenzymes. Finally, the use of Ca2+ inhibitors (LaCl3 and EDTA) and deletion of spcF (a calmodulin-like protein gene) further demonstrated that the overexpression of those genes was regulated via calcium signaling in B. subtilis subjected to GO stress. IMPORTANCE To effectively convert lignocellulose into fermentable sugars, high lignocellulolytic enzyme loading is needed. Graphene oxide (GO) has been shown to promote exoenzyme (lignocellulolytic enzyme, amylase, peptidase, and protease) production in some microorganisms; however, the regulatory mechanism of the biosynthesis of lignocellulolytic enzymes under GO stress remains unclear. In this work, the lignocellulolytic enzyme production of B. subtilis under GO stress was investigated, and the potential mechanism by which B. subtilis enhanced lignocellulolytic enzyme production through the calcium signaling pathway under GO stress was proposed. This work revealed the role of calcium signaling in the production of enzymes under external environmental stress and provided a direction to facilitate lignocellulolytic enzyme production by B. subtilis.

Preparation of bimetal-polydopamine organic frameworks with core-shell structure and their application in HER2 detection.

Human epidermal growth factor receptor 2 (HER2) is one of the specific markers of breast cancer, which is of great significance to the early diagnosis and prognosis of breast cancer. Here, a fluorescence biosensor was established to detect HER2 based on the fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) occurring between the bimetal-polydopamine organic framework with core-shell structure Au@PDA@UiO-66 and the Cy5 fluorophore in HER2-Cy5-Apt. Au@PDA@UiO-66 owns high-efficiency fluorescence quenching ability due to its large specific surface area and strong adsorption of single-stranded DNA. When the target appears, the fluorescence recovery space mediated by the target is large, so the proposed biosensor has better sensitivity in theory. Under optimized conditions, the proposed fluorescent biosensor can detect HER2 in a range of 0.005 ng mL-1 to 15 ng mL-1, with an actual detection limit as low as 0.005 ng mL-1. Corresponding selective experiments, reproducible experiments, and spiked experiments performed well, showing its great potential in HER2 detection.