High Background Signal Research Articles

Programmable Entropy-Driven Circuit-Cascaded Self-Feedback DNAzyme Network for Ultra-Sensitive Fluorescence and Photoelectrochemical Dual-Mode Biosensing.

Inspired by natural DNA networks, programmable artificial DNA networks have become an attractive tool for developing high-performance biosensors. However, there is still a lot of room for expansion in terms of sensitivity, atom economy, and result self-validation for current microRNA sensors. In this protocol, miRNA-122 as a target model, an ultrasensitive fluorescence (FL) and photoelectrochemical (PEC) dual-mode biosensing platform is developed using a programmable entropy-driven circuit (EDC) cascaded self-feedback DNAzyme network. The well-designed EDC realizes full utilization of the DNA strands and improves the atomic economy of the signal amplification system. The unique and rational design of the double-CdSe quantum-dot-released EDC substrate and the cascaded self-feedback DNAzyme amplification network significantly avoids high background signals and enhances sensitivity and specificity. Also, the enzyme-free, programmable EDC cascaded DNAzyme network effectively avoids the risk of signal leakage and enhances the accuracy of the sensor. Moreover, the introduction of superparamagnetic Fe3O4@SiO2-cDNA accelerates the rapid extraction of E2-CdSe QDs and E3-CdSe QDs, which greatly improves the timeliness of sensor signal reading. In addition to the strengths of linear range (6 orders of magnitude) and stability, the biosensor design with dual signal reading makes the test results self-confirming.

High ODMR contrast and alignment of NV centers in microstructures grown on heteroepitaxial diamonds

Heteroepitaxial chemical vapor deposition is the most promising option to fabricate wafer-scale monocrystalline diamonds for quantum applications. Previously, we demonstrated the feasibility to manufacture functional micrometer-sized pyramids on as-grown heteroepitaxial diamond as well as their quantum optical characteristics. Due to high background signals and microfabrication challenges, these pyramids could not compete with homoepitaxially grown structures. In this study, we overcame these problems with a nominally undoped buffer layer between the heteroepitaxial substrate and the pyramidal microstructure to reduce the signal-to-noise ratio from the substrate on the spin measurements of the nitrogen-vacancy (NV) center. Moreover, the microfabrication was improved to reach a higher angle of the pyramidal side plane, corresponding to the {111} facets. These improvements lead to pyramids on which each facet contains almost purely only one of the four possible NV orientations as shown by optically detected magnetic resonance (ODMR). ODMR shows a very high contrast of 19% without an external magnet and of 13% for a single spin resonance in the presence of a magnetic field. The contrast is more than doubled compared to our previous study. The T2* dephasing time of the NV centers of the samples ranges from 0.02 to 0.16 μs. The P1 center is a single substitutional nitrogen center, and the P1 densities range from 1.8 to 5 ppm.

Synthesis and Evaluation of [18F]AlF-NOTA-c-DVAP: A Novel PET Probe for Imaging GRP78 in Cancer.

GRP78, a member of the HSP70 superfamily, is an endoplasmic reticulum chaperone protein overexpressed in various cancers, making it a promising target for cancer imaging and therapy. Positron emission tomography (PET) imaging offers unique advantages in real time, noninvasive tumor imaging, rendering it a suitable tool for targeting GRP78 in tumor imaging to guide targeted therapy. Several studies have reported successful tumor imaging using PET probes targeting GRP78. However, existing PET probes face challenges such as low tumor uptake, inadequate in vivo distribution, and high abdominal background signal. Therefore, this study introduces a novel peptide PET probe, [18F]AlF-NOTA-c-DVAP, for targeted tumor imaging of GRP78. [18F]AlF-NOTA-c-DVAP was radiolabeled with fluoride-18 using the aluminum-[18F]fluoride ([18F]AlF) method. The study assessed the partition coefficients, stability in vitro, and metabolic stability of [18F]AlF-NOTA-c-DVAP. Micro-PET imaging, pharmacokinetic analysis, and biodistribution studies were carried out in tumor-bearing mice to evaluate the probe's performance. Docking studies and pharmacokinetic analyses of [18F]AlF-NOTA-c-DVAP were also performed. Immunohistochemical and immunofluorescence analyses were conducted to confirm GRP78 expression in tumor tissues. The probe's binding affinity to GRP78 was analyzed by molecular docking simulation. [18F]AlF-NOTA-c-DVAP was radiolabeled in just 25 min with a high yield of 51 ± 16%, a radiochemical purity of 99%, and molar activity within the range of 20-50 GBq/μmol. [18F]AlF-NOTA-c-DVAP demonstrated high stability in vitro and in vivo, with a logD value of -3.41 ± 0.03. Dynamic PET imaging of [18F]AlF-NOTA-c-DVAP in tumors showed rapid uptake and sustained retention, with minimal background uptake. Biodistribution studies revealed rapid blood clearance and excretion through the kidneys following a single-compartment reversible metabolic model. In PET imaging, the T/M ratios for A549 tumors (high GRP78 expression), MDA-MB-231 tumors (medium expression), and HepG2 tumors (low expression) at 60 min postintravenous injection were 10.48 ± 1.39, 6.25 ± 0.47, and 3.15 ± 1.15% ID/g, respectively, indicating a positive correlation with GRP78 expression. This study demonstrates the feasibility of using [18F]AlF-NOTA-c-DVAP as a PET tracer for imaging GRP78 in tumors. The probe shows promising results in terms of stability, specificity, and tumor targeting. Further research may explore the clinical utility and potential therapeutic applications of this PET tracer for cancer diagnosis.

Pimonidazole-alkyne conjugate for sensitive detection of hypoxia by Cu-catalyzed click reaction

Hypoxia is involved in various diseases, such as cancers. Pimonidazole has often been used as the gold-standard marker to visualize hypoxic regions. Pimonidazole labels hypoxic regions by forming a covalent bond with a neighboring protein under hypoxic conditions in the body, which is detected by immunohistochemistry performed on tissue sections. To date, some pimonidazole-fluorophore conjugates have been reported as fluorescent probes for hypoxia imaging that do not require immunostaining. They are superior to pimonidazole because immunostaining can produce high background signals. However, large fluorophores in the conjugates may alter the original biodistribution and reactivity. Here, we report a new hypoxia marker, Pimo-yne, as a pimonidazole-alkyne conjugate. Pimo-yne has a similar hypoxia detection capability as pimonidazole because the alkyne tag is small and can be detected by Cu-catalyzed click reaction with azide-tagged fluorescent dyes. We successfully visualized hypoxic regions in tumor tissue sections using Pimo-yne with reduced background signals. The detected regions overlapped well with those detected by pimonidazole immunohistochemistry. To further reduce the background, we employed a turn-on azide-tagged fluorescent dye.Graphical abstract

Trace Silicon Determination in Biological Samples by Inductively Coupled Plasma Mass Spectrometry (ICP-MS): Insight into the Volatility of Silicon Species in Hydrofluoric Acid Digests for Optimal Sample Preparation and Introduction to ICP-MS

A method for the determination of trace levels of silicon from biological materials by inductively coupled plasma mass spectrometry (ICP-MS) has been developed. The volatility of water-soluble silicon species, hexafluorosilicic acid (H2SiF6), and sodium metasilicate (Na2SiO3) was investigated by evaporating respective solutions (50 µg/mL silicon) in HNO3), nitric acid + hydrochloric acid (HNO3 + HCl), and nitric acid + hydrochloric acid + hydrofluoric acid (HNO3 + HCl + HF) at 120 °C on a hot-block to near dryness. The loss of silicon from H2SiF6 solutions was substantial (>99%) regardless of the digestion medium. Losses were also substantial (>98%) for metasilicate solutions heated in HNO3 + HCl + HF, while no significant loss occurred in HNO3 or HNO3 + HCl. These results show that H2SiF6 species were highly volatile and potential losses could confound accuracy at trace level determinations by ICP-MS if digestates prepared in HF are heated to eliminate HF. Among the various matrices comprising major elements, sodium appeared to be effective in reducing silicon loss from H2SiF6 solutions. Excess sodium chloride (NaCl) matrix provided better stability, improving silicon recoveries by up to about 80% in evaporated HF digestates of soil and mine waste samples, but losses could not be fully prevented. To safely remove excess acids and circumvent the adverse effects of excess HF (e.g., risk of high Si background signals), a two-step digestion scheme was adopted for the preparation of biological samples containing trace silicon levels. A closed-vessel digestion was performed either in 4 mL of concentrated HNO3 and 1 mL of concentrated HCl or 4 mL of concentrated HNO3, 1 mL of concentrated HCl and 1 mL of concentrated HClO4 on a hot plate at 140 °C. Digestates were then evaporated to incipient dryness at 120 °C to remove the acids. A second closed-vessel digestion was carried out to dissolve silicates in 0.5 mL of concentrated HNO3 and 0.5 mL of concentrated HF at 130 °C. After digestion, digestates were diluted to 10 mL. The solution containing about 5% HNO3 and 5% HF was directly analyzed by ICP-MS equipped with an HF-inert sample introduction system. The limit of detection was about 110 µg/L for 28Si when using the Kinetic Energy Discrimination (KED) mode. The method was used to determine silicon in various plant and tissue certified reference materials. Data were acquired for 28Si using KED and standard (STD) modes, and 74Ge and 103Rh as internal standard elements. There was not any significant difference between the accuracy and precision of the results obtained with 74Ge and 103Rh within the same measurement mode. Precision, calculated as relative standard deviation for four replicate analyses, varied from 5.3 (tomato leaves) to 21% (peach leaves) for plant and from 2.2 (oyster tissue) to 33% (bovine liver) for tissue SRM/CRMs. Poor precision was attributed to material heterogeneity and the large particle size distribution. An analysis of lung tissue samples from those with occupational exposure to silica dust revealed that tissues possessed substantial levels of water-soluble silicates, but the most silicon was present in the particulate matter fraction.

A Point-of-Care Nucleic Acid Quantification Method by Counting Light Spots Formed by LAMP Amplicons on a Paper Membrane.

Nucleic acid quantification, allowing us to accurately know the copy number of target nucleic acids, is significant for diagnosis, food safety, agricultural production, and environmental protection. However, current digital quantification methods require expensive instruments or complicated microfluidic chips, making it difficult to popularize in the point-of-care detection. Paper is an inexpensive and readily available material. In this study, we propose a simple and cost-effective paper membrane-based digital loop-mediated isothermal amplification (LAMP) method for nucleic acid quantification. In the presence of DNA fluorescence dyes, the high background signals will cover up the amplicons-formed bright spots. To reduce the background fluorescence signals, a quencher-fluorophore duplex was introduced in LAMP primers to replace non-specific fluorescence dyes. After that, the amplicons-formed spots on the paper membrane can be observed; thus, the target DNA can be quantified by counting the spots. Take Vibrio parahaemolyticus DNA detection as an instance, a good linear relationship is obtained between the light spots and the copy numbers of DNA. The paper membrane-based digital LAMP detection can detect 100 copies target DNA per reaction within 30 min. Overall, the proposed nucleic acid quantification method has the advantages of a simple workflow, short sample-in and answer-out time, low cost, and high signal-to-noise, which is promising for application in resourced limited areas.

An ultrasensitive smartphone-assisted bicolor-ratiometric fluorescence sensing platform based on a “noise purifier” for point-of-care testing of pathogenic bacteria in food

Non-specific binding in fluorescence resonance energy transfer (FRET) remains a challenge in foodborne pathogen detection, resulting in interference of high background signals. Herein, we innovatively reported a dual-mode FRET sensor based on a “noise purifier” for the ultrasensitive quantification of Escherichia coli O157:H7 in food. An efficient FRET system was constructed with polymyxin B-modified nitrogen-sulfur co-doped graphene quantum dots (N, S-GQDs@PMB) as donors and aptamer-modified yellow carbon dots (Y-CDs@Apt) as acceptors. Magnetic multi-walled carbon nanotubes (Fe@MWCNTs) were employed as a “noise purifier” to reduce the interference of the fluorescence background. Under the background purification mode, the sensitivity of the dual-mode signals of the FRET sensor has increased by an order of magnitude. Additionally, smartphone-assisted colorimetric analysis enabled point-of-care detection of E. coli O157:H7 in real samples. The developed sensing platform based on a “noise purifier” provides a promising method for ultrasensitive on-site testing of trace pathogenic bacteria in various foodstuffs.

Metal–organic frameworks with aggregation‐induced emission

AbstractAggregation‐induced emission (AIE) materials exhibit remarkable emission in the aggregated or solid state while demonstrating minimal emission in dilute solutions. In contrast to conventional luminescent materials, AIE luminogens (AIEgens) offer several advantages in the aggregate state, including high quantum yield, excellent photostability, and low background signals, making them highly promising for diverse applications. Integrating AIEgens into designable metal–organic frameworks (MOFs) enables tunable and well‐ordered AIE materials, allowing for precise control over photophysical properties and deeper exploration of AIE mechanisms. Numerous AIE MOFs have been constructed and investigated, and several reviews focus on their structure design and applications in sensing and bioimaging. This review highlights the state‐of‐the‐art advancements in AIE MOFs, including mechanisms, design strategies, and applications in chemical sensing, bioimaging, and disease therapy. The challenges associated with practical applications of AIE MOFs are also addressed, with an emphasis on their large‐scale production involved interdisciplinary collaboration. This comprehensive review aims at guiding further development of AIE MOFs and promoting their practical applications in analysis, healthcare, and other luminescence related fields.

Fluorogenic CRISPR for genomic DNA imaging

Genomic DNA exhibits high heterogeneity in terms of its dynamic within the nucleus, its structure and functional roles. CRISPR-based imaging approaches can image genomic loci in living cells. However, conventional CRISPR-based tools involve expressing constitutively fluorescent proteins, resulting in high background and nonspecific nucleolar signal. Here, we construct fluorogenic CRISPR (fCRISPR) to overcome these issues. fCRISPR is designed with dCas9, an engineered sgRNA, and a fluorogenic protein. Fluorogenic proteins are degraded unless they are bound to specific RNA hairpins. These hairpins are inserted into sgRNA, resulting in dCas9: sgRNA: fluorogenic protein ternary complexes that enable fluorogenic DNA imaging. With fCRISPR, we image various genomic DNA in different human cells with high signal-to-noise ratio and sensitivity. Furthermore, fCRISPR tracks chromosomes dynamics and length. fCRISPR also allows DNA double-strand breaks (DSBs) and repair to be tracked in real time. Taken together, fCRISPR offers a high-contrast and sensitive platform for imaging genomic loci.

ZIF-67 Anchored on MoS2/rGO Heterostructure for Non-Enzymatic and Visible-Light-Sensitive Photoelectrochemical Biosensing.

Graphene and graphene-like two-dimensional layered nanomaterials-based photoelectrochemical (PEC) biosensors have recently grown rapidly in popularity thanks to their advantages of high sensitivity and low background signal, which have attracted tremendous attention in ultrahigh sensitive small molecule detection. This work proposes a non-enzymatic and visible-light-sensitive PEC biosensing platform based on ZIF-67@MoS2/rGO composite which is synthesized through a facile and one-step microwave-assisted hydrothermal method. The combination of MoS2 and rGO could construct van der Waals heterostructures, which not only act as visible-light-active nanomaterials, but facilitate charge carriers transfer between the photoelectrode and glassy carbon electrode (GCE). ZIF-67 anchored on MoS2/rGO heterostructures provides large specific surface areas and a high proportion of catalytic sites, which cooperate with MoS2 nanosheets, realizing rapid and efficient enzyme-free electrocatalytic oxidation of glucose. The ZIF-67@MoS2/rGO-modified GCE can realize the rapid and sensitive detection of glucose at low detection voltage, which exhibits a high sensitivity of 12.62 μAmM-1cm-2. Finally, the ZIF-67@MoS2/rGO PEC biosensor is developed by integrating the ZIF-67@MoS2/rGO with a screen-printed electrode (SPE), which exhibits a high sensitivity of 3.479 μAmM-1cm-2 and a low detection limit of 1.39 μM. The biosensor's selectivity, stability, and repeatability are systematically investigated, and its practicability is evaluated by detecting clinical serum samples.

OSL at elevated temperature of smart chip cards for retrospective dosimetry

Smart chip cards are a promising material in retrospective dosimetry due to their high collectability with low replacement cost. In this study, dose reconstruction using OSL at elevated temperatures for various chip cards was investigated. The approximate spectral emission of the chip cards was determined through a comparison of radiation-induced and intrinsic background TL signals using different detection filters. In the OSL decay curve, a fast component (0.0–0.4 s) was integrated to avoid high intrinsic background signals. To minimize sensitivity changes due to heat treatment, a protocol to measure OSL at 100 °C was developed and the data were compared with those from a similar protocol for OSL measured at room temperature. In the 0.1 - 5 Gy range, linear and power functions were fitted for room temperature and 100 °C OSL protocols, respectively. The minimum detectable dose was evaluated immediately after exposure, with values of 25 mGy for room temperature and 240 mGy for 100 °C OSL being determined. Signal fading was measured up to 30 days after irradiation for the investigated protocols. A dose recovery test was made 10 days after irradiation using various chip cards, and the 100 °C OSL protocol produced more reliable results than the room temperature OSL protocol. As a result, the study emphasizes the necessity of further investigation of OSL at high temperatures using chip cards.

Development of a one-pot and sequence-specific LAMP assay based on the self-quenching probe integrated by the complementary oligonucleotide for an enhanced fluorescence quenching

Single-modified fluorogenic primer (Sfp) enables accurate identification of LAMP amplicons without being affected by non-specific products. However, the fluorescence self-quenching by nucleobases for Sfp is generally of low efficiency, and the high background signal makes it a great challenge to achieve visual inspection with naked eyes. In the present study, the oligonucleotide (Ao) complementary to Sfp was designed, which would hybridize to Sfp and dramatically heighten the quenching effect, leading to a low background signal in negative reaction. Instead, for positive reaction, Sfp is incorporated into the double-stranded amplicons, resulting in dequenching and consequently, enhanced fluorescence. The detection scheme can be further improved by a dual-color fluorescence strategy, allowing visual detection of 1 pg rainbow trout DNA in a closed-tube format within 30 min. Therefore, our LAMP-Ao-Sfp assay represents a useful tool for rapid and sensitive detection, and can serve as a reliable method for on-site detection in low-resource settings.

Dephosphorylation Switch DNAzyme-RCA Circuit: A Robust Strategy for the Homogeneous and Reliable Detection of FTO Demethylase.

Fat mass and obesity-associated protein (FTO) plays a crucial role in regulating the dynamic modification of N6-methyladenosine (m6A) in eukaryotic mRNA. Sensitive detection of the FTO level and efficient evaluation of the FTO demethylase activity are of great importance to early cancer diagnosis and anticancer drug discovery, which are currently challenged by limited sensitivity/precision and low throughput. Herein, a robust strategy based on the dephosphorylation switch DNAzyme-rolling circle amplification (RCA) circuit, termed DSD-RCA, is developed for highly sensitive detection of FTO and inhibitor screening. Initially, the catalytic activity of DNAzyme is silenced by engineering with an m6A modification in its catalytic core. Only in the presence of target FTO can the methyl group on DNAzyme be eliminated, resulting in the activation of the catalytic activity of DNAzyme and thus cleaving the hairpin substrate to release numerous primers. Different from the conventional methods that use the downstream cleavage primer with the original 3'-hydroxyl end directly as the RCA primer with the problem of high background signal, which should be compensated by additional separation and wash steps in heterogeneous format, our DSD-RCA assay uses the upstream cleavage primer with a 2',3'-cyclic phosphate terminus at the 3'-end serving as an intrinsically blocked 3' end. Only after a dephosphorylation reaction mediated by T4 polynucleotide kinase can the upstream cleavage primers with a resultant 3'-hydroxyl end be extended by RCA. With the high signal-to-noise ratio and homogeneous property, the proposed platform can sensitively detect FTO with a limit of detection of 31.4 pM, and the relative standard deviations (RSDs %) ranging from 0.8 to 2.0% were much lower than the heterogeneous methods. The DSD-RCA method was applied for analyzing FTO in cytoplasmic lysates from different cell lines and tissues of breast cancer patients and further used for screening FTO inhibitors without the need for separation or cleaning, providing an opportunity for achieving high throughput and demonstrating the potential applications of this strategy in disease diagnostics, drug discovery, and biological applications.

Semi-Quantitative Analyses of Ferromanganese Nodules from the Pacific Ocean Using Synchrotron X-ray Powder Diffraction

The geochemistry and mineralogy of Mn nodules offer crucial insights into the origins, environmental changes, and distribution of abyssal resources. However, the conventional laboratory X-ray diffractometer, usually employed for semi-quantitative analysis of mineral composition in Mn nodules, often fails to sufficiently detect minor phases due to beam flux limitations and high background signals. In this study, we investigated differences in manganate composition, even when comprising around 1% of the phase fraction, in two manganese nodules (KC-8 and KODOS-10) using high-resolution synchrotron X-ray diffraction. The Mn/Fe ratios of KC-8 and KODOS-10 were 1.32 and 6.24, respectively, indicating that KC-8 and KODOS-10 were predominantly formed in hydrogenetic and diagenetic environments. Both samples contained quartz, vernadite, buserite, and feldspar. Todorokite and illite were exclusively observed in KODOS-10. In KC-8, the phase fractions of vernadite and buserite among manganates ranged from 94(5)%–100(4)% and 6(1)%–0%, respectively. However, in KODOS-10, the fractions of vernadite, buserite, and todorokite ranged from 47(1)%–56(2)%, 33.6(4)%–40.1(3)%, and 10(3)%–16.3(8)%, respectively.

Performance of a Modified ALLType NGS HLA Typing Assay with the Ion Chef/Ion S5/TypeStream Visual System

Abstract OBJECTIVE S: The commercial ALLType assay (One Lambda, West Hills, CA) enables accurate, high-resolution typing of 11 HLA genes using the Ion Chef/Ion S5/TypeStream Visual (TSV) system. The assay features a multiplexed PCR to enrich all target HLA genes in a sample, and up to 48 samples can be indexed and sequenced in a run. The assay is laborious due to the late pooling of indexed libraries. Early pooling significantly simplifies the workflow but may increase the background noise and typing errors. This project aims to examine the feasibility of early pooling immediately after the indexing step and evaluate the performance of such a modified ALLType protocol. METHODS A total of 75 unique samples with diverse HLA genotypes were included in this study, which comprised 52 proficiency testing samples of three different sources, 10 stem cell recipients, 7 stem cell donors (including 2 buccal swabs), and 6 deceased organ donors. Genomic DNA extracted from these samples was tested in four sequencing runs, with 15-31 samples per run, using the modified ALLType procedure (reagent lot: 15). The results were compared with the reference typings generated by the manufacturer-established protocol. All data were analyzed using TSV (version 3.0) and the IPD-IMGT/HLA library (version 3.47); the default analysis parameters were applied. The primary endpoint of the study was the concordance of 3-field typing results. The secondary endpoint was key health metrics, including allele balance, key-exon coverage, and background noise. We also evaluated the saving on hands-on time and reagents with the modified protocol. RESULTS The concordance rates of 3-field typing results were 100% for HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, and HLA-DRB1, and 96.2% for HLA-DRB3/4/5. The three discrepant HLA-DRB3/4/5 results with the modified protocol were false positive allele calls due to a small number of contaminating reads, which were corrected by increasing the threshold for HLA-DRB3/4/5 in TSV. The allele balance and exon 2 coverage across all samples and genes were comparable between the modified and reference protocols, with R-squared values of 0.83 and 0.81, respectively. High background signals in exons were observed in only 0.6% of all samples with both protocols. The modified protocol reduced the hands-on time by 15%, from 6.5 to 5.5 hours, and reduced the use of library preparation reagents by 30%. CONCLUSION It is feasible to pool indexed libraries early during the library preparation process without increasing the background noise in the sequencing data. The modified ALLType protocol showed typing accuracy and health metrics comparable to the original protocol, with significant savings on time and reagents.

Thermal stability and micrdose-based coupling CRISPR/Cas12a biosensor for amplification-free detection of hgcA gene in paddy soil

The lack of point-of-use (POU) methods hinders the utilization of the hgcA gene to rapidly evaluate methylmercury risks. CRISPR/Cas12a is a promising technology, but shortcomings such as low sensitivity, a strict reaction temperature and high background signal limit its further utilization. Here, a thermally stable microsystem-based CRISPR/Cas12a biosensor was constructed to achieve POU analysis for hgcA. First, three target gRNAs were designed to recognize hgcA. Then, a microsystem was developed to eliminate the background signal. Next, the effect of temperature on the activity of the Cas12a-gRNA complex was explored and its thermal stability was discovered. After that, coupling gRNA assay was introduced to improve sensitivity, exhibiting a limit of detection as low as 0.49 pM with a linear range of 0.98–125 pM, and a recovery rate between 90 and 110 % for hgcA. The biosensor was finally utilized to assess hgcA abundance in paddy soil, and high abundance of hgcA was found in these paddy soil samples. This study not only systematically explored the influence of temperature and microsystem on CRISPR/Cas12a, providing vital references for other novel CRISPR-based detection methods, but also applied the CRISPR-based analytical method to the field of environmental geochemistry for the first time, demonstrating enormous potential for POU detection in this field.

Aggregation-induced emission for the detection of peptide ligases with improving ligation efficiency

BackgroundMonitoring peptide ligase activity is of great significance for biological research, medical diagnosis, and drug discovery. The current methods for the detection of peptide ligases suffer from the limitations of high background signal, elaborate design of substrate, and high reversibility of ligation reaction. In this work, we proposed a simple and sensitive method for ligase detection with reducing ligation reversibility on the basis of aggregation-induced emission (AIE) mechanism. ResultsThe peptide probes labeled with AIE luminogens (AIEgens) were water-soluble and emitted weak fluorescence. After ligation reaction, the enzymatic products with AIEgens showed high hydrophobicity and could readily assembly into aggregates, thus lighting up the fluorescence. More interestingly, the formation of aggregates pushed the equilibrium to the generation of the desired ligation products, thus improving the catalytic efficiency by driving the reaction towards completion. The ligation reaction conversion rate (>80 %) is significantly higher than that without blocking the reversibility with additional treatment. With sortase A (SrtA) as the analyte example, the detection limit of this method was found to be 0.01 nM with a linear range of 0–50 nM. The system was applied to evaluate the inhibition efficiency of berberine chloride and quercetin and determine the activity of SrtA in serum, lysate and Staphylococcus aureus with satisfactory results. SignificanceThis study indicated that the ligation efficiency and detection sensitivity can be improved by reducing ligation reversibility through AIE phenomenon. The proposed strategy could be used for the detection of other peptide ligases by adopting sequence-specific peptide substrates.

Targeted delivery of cathepsin-activatable near-infrared fluorescence probe for ultrahigh specific imaging of peritoneal metastasis

Inadequate cytoreductive surgery (CRS) has been identified as a prognostic factor for poor patient outcomes in cases of peritoneal metastasis. While imaging probes are used to identify peritoneal metastasis to facilitate CRS, many of these probes exhibit high background signals, resulting in a significant delay in achieving a satisfactory tumor-to-normal ratio (TNR) due to prolonged clearance time. In this study, we designed a novel fluorescent probe named Tras-AA-Cy NH2, which enables the relatively rapid imaging of subcutaneous tumors and peritoneal tumors while maintaining a high TNR. Mechanistically, Tras-AA-Cy NH2 exhibits selective targeting towards the Human epidermal growth factor receptor 2 on the surface of cancer cells. Following internalization, it undergoes enzymatic cleavage catalyzed by the overexpressed cathepsin, leading to the subsequent release of near-infrared fluorophores. Consequently, Tras-AA-Cy NH2 achieved a TNR of 7.8 at 6 h and 21.4 at 24 h in subcutaneous tumor mice. Even after 522 h of in vivo circulation, the TNR remained above 5, indicating an ultralong imaging time window. It is noteworthy that Tras-AA-Cy NH2 has demonstrated successful utilization for peritoneal tumor-specific imaging and further affirmed its tumor tissue-specific recognition capability using human resected tissues. In summary, these findings underscore the rational design of Tras-AA-Cy NH2 for visualizing peritoneal tumors.

Target-Induced Photocurrent-Polarity-Switching PEC Sensing Platform Based on In Situ Generation of Oxygen Vacancy-Modulated Energy Band Structures.

The polarity of the photocurrent can be modulated by tunable bipolar photoelectrochemical (PEC) behavior, which is anticipated to address the issues of high background signal caused by traditional unidirectional increasing/decreasing response and false-positive/false-negative problems. Here, a new approach is suggested for the first time, which employs a target-induced enzyme-catalyzed reaction and in situ oxygen vacancy (OV) generation to achieve heterojunction photocurrent switching for highly sensitive detection of alkaline phosphatase (ALP). Among them, the ALP can catalyze the decomposition of ascorbic acid phosphate to produce ascorbic acid, which not only acts as an electron donor to change the redox environment but also acts as a reducing agent to introduce OVs into BiOBr semiconductors in cooperation with illumination. The introduction of vacancies can effectively modulate the energy band structure of BiOBr, while with the change of redox conditions, the transfer path of photogenerated carriers is changed, thus realizing the switching of photocurrents, which leads to its use in the construction of a negative-background anti-interference PEC sensing platform, achieving a wide linear range from 0.005 to 500 U·L-1 with a low detection limit of 0.0017 U·L-1. In conclusion, the photocurrent switching operation of this system is jointly regulated by chemistry, optics, and carrier motion, which provides a new idea for the construction of a PEC sensing platform based on photocurrent polarity switching.

Fibrillization Process of Human Amyloid-Beta Protein (1-40) under a Molecular Crowding Environment Mimicking the Interior of Living Cells Using Cell Debris.

Molecular crowding environments play a crucial role in understanding the mechanisms of biological reactions. Inside living cells, a diverse array of molecules coexists within a volume fraction ranging from 10% to 30% v/v. However, conventional spectroscopic methods often face difficulties in selectively observing the structures of particular proteins or membranes within such molecularly crowded environments due to the presence of high background signals. Therefore, it is crucial to establish in vitro measurement conditions that closely resemble the intracellular environment. Meanwhile, the neutron scattering method offers a significant advantage in selectively observing target biological components, even within crowded environments. Recently, we have demonstrated a novel scattering method capable of selectively detecting the structures of targeted proteins or membranes in a closely mimicking intracellular milieu achieved utilizing whole-cell contents (deuterated-cell debris). This method relies on the inverse contrast matching technique in neutron scattering. By employing this method, we successfully observed the fibrillization process of human amyloid beta-protein (Aβ 1-40) under a molecular crowding environment (13.1% w/v cell debris, Aβ/cell debris = ~1/25 w/w) that closely mimics the interior of living cells. Aβ protein is well known as a major pathogenic component of Alzheimer's disease. The present results combining model simulation analyses clearly show that the intracellular environment facilitates the potential formation of even more intricate higher-order aggregates of Aβ proteins than those previously reported.