Colorimetric Immunoassays with Boronic Acid-Decorated, Peroxidase-like Metal-Organic Frameworks as the Carriers of Antibodies and Enzymes.

The sensitivity of immunoassays is generally limited by the low signal reporter/recognition element ratio. Nanomaterials serving as the carriers can enhance the loading number of signal reporters, thus improving the detection sensitivity. However, the general immobilization strategies, including direct physical adsorption and covalent coupling, may cause the random orientation and conformational change in proteins, partially or completely suppressing the enzymatic activity and the molecular recognition ability. In this work, we proposed a strategy to load recognition elements of antibodies and enzyme labels using boronic acid-modified metal-organic frameworks (MOFs) as the nanocarriers for signal amplification. The conjugation strategy was proposed based on the boronate ester interactions between the carbohydrate moieties in antibodies and enzymes and the boronic acid moieties on MOFs. Both enzymes and MOFs could catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2, therefore achieving dual signal amplification. To indicate the feasibility and sensitivity of the strategy, colorimetric immunoassays of prostate specific antigen (PSA) were performed with boronic acid-modified Cu-MOFs as peroxidase mimics to catalyze TMB oxidation and nanocarriers to load antibody and enzyme (horseradish peroxidase, HRP). According to the change in the absorbance intensity of the oxidized TMB (oxTMB), PSA at the concentration range of 1~250 pg/mL could be readily determined. In addition, this work presented a site-specific and oriented conjugation strategy for the modification of nanolabels with recognition elements and signal reporters, which should be valuable for the design of novel biosensors with high sensitivity and selectivity.

Optical Bioassays Based on the Signal Amplification of Redox Cycling.

Optical bioassays are challenged by the growing requirements of sensitivity and simplicity. Recent developments in the combination of redox cycling with different optical methods for signal amplification have proven to have tremendous potential for improving analytical performances. In this review, we summarized the advances in optical bioassays based on the signal amplification of redox cycling, including colorimetry, fluorescence, surface-enhanced Raman scattering, chemiluminescence, and electrochemiluminescence. Furthermore, this review highlighted the general principles to effectively couple redox cycling with optical bioassays, and particular attention was focused on current challenges and future opportunities.

Simultaneous detection of breast cancer biomarkers circROBO1 and BRCA1 based on a CRISPR-Cas13a/Cas12a system.

Breast cancer is reported to be one of the most lethal cancers in women, and its multi-target detection can help improve the accuracy of diagnosis. In this work, a cluster regularly interspaced short palindromic repeats (CRISPR)-Cas13a/Cas12a-based system was established for the simultaneous fluorescence detection of breast cancer biomarkers circROBO1 and BRCA1. CRISPR-Cas13a and CRISPR-Cas12a were directly activated by their respective targets, resulting in the cleavage of short RNA and DNA reporters, respectively, thus the signals of 6-carboxyfluorescein (FAM) and 6-carboxy-xrhodamine (ROX) were restored. As the fluorescence intensities of FAM and ROX were dependent on the concentrations of circROBO1 and BRCA1, respectively, synchronous fluorescence scanning could achieve one-step detection of circROBO1 and BRCA1 with detection limits of 0.013 pM and 0.26 pM, respectively. The system was highly sensitive and specific, holding high diagnostic potential for the detection of clinical samples. Furthermore, the competing endogenous RNA mechanism between circROBO1 and BRCA1 was also explored, providing a reliable basis for the intrinsic regulatory mechanism of breast cancer.

DNA tetrahedron-based dual-signal fluorescence detection of apoE4 gene sites on a microplate reader.

As a neurodegenerative disorder, Alzheimer's disease (AD) is characterized by cognitive dysfunction and behavioral impairment. Among the various genetic risk factors for AD, apoE4 gene plays a pivotal role in the onset and progression of AD, and detection of apoE4 gene holds significance for prevention and early diagnosis of AD. Herein, dual-signal fluorescence detection of fragments associated with apoE ε4 allele near codon 112 (Tc1) and codon 158 (Tc2) was achieved using DNA tetrahedron nanostructure (DTN). The Förster resonance energy transfer (FRET) process in the DTN was initiated in which the nucleic acid intercalating dye thiazole orange (TO) served as the donor and the cyanine dyes of cyanine3 (Cy3) and cyanine5 (Cy5) at the two vertices of DTN served as the acceptors. In the presence of Tc1 and Tc2, the FRET process between TO and the cyanine dyes was hindered by the enzymatic cleavage reaction, which ensures the dual-signal fluorescence assay of apoE4 gene sites. The limit of detection for Tc1 and Tc2 was estimated to be 0.82 nM and 0.77 nM, respectively, and the whole assay was accomplished within 1 h on a microplate reader. The proposed method thus possesses the advantages of easy operation, short detection time, and high-throughput capability.

Assessing base-resolution DNA mechanics on the genome scale.

Intrinsic DNA properties including bending play a crucial role in diverse biological systems. A recent advance in a high-throughput technology called loop-seq makes it possible to determine the bendability of hundred thousand 50-bp DNA duplexes in one experiment. However, it's still challenging to assess base-resolution sequence bendability in large genomes such as human, which requires thousands of such experiments. Here, we introduce 'BendNet'-a deep neural network to predict the intrinsic DNA bending at base-resolution by using loop-seq results in yeast as training data. BendNet can predict the DNA bendability of any given sequence from different species with high accuracy. To explore the utility of BendNet, we applied it to the human genome and observed DNA bendability is associated with chromatin features and disease risk regions involving transcription/enhancer regulation, DNA replication, transcription factor binding and extrachromosomal circular DNA generation. These findings expand our understanding on DNA mechanics and its association with transcription regulation in mammals. Lastly, we built a comprehensive resource of genomic DNA bendability profiles for 307 species by applying BendNet, and provided an online tool to assess the bendability of user-specified DNA sequences (http://www.dnabendnet.com/).

The intratumor mycobiome promotes lung cancer progression via myeloid-derived suppressor cells.

Although polymorphic microbiomes have emerged as hallmarks of cancer, far less is known about the role of the intratumor mycobiome as living microorganisms in cancer progression. Here, using fungi-enriched DNA extraction and deep shotgun metagenomic sequencing, we have identified enriched tumor-resident Aspergillus sydowii in patients with lung adenocarcinoma (LUAD). By three different syngeneic lung cancer mice models, we find that A. sydowii promotes lung tumor progression via IL-1ß-mediated expansion and activation of MDSCs, resulting in suppressed activity of cytotoxic T lymphocyte cells and accumulation of PD-1+ CD8+ T cells. This is mediated by IL-1ß secretion via ß-glucan/Dectin-1/CARD9 pathway. Analysis of human samples confirms that enriched A. sydowii is associated with immunosuppression and poor patient outcome. Our findings suggest that intratumor mycobiome, albeit at low biomass, promotes lung cancer progression and could be targeted at the strain level to improve patients with LUAD outcome.

Overview on the Development of Alkaline-Phosphatase-Linked Optical Immunoassays.

The drive to achieve ultrasensitive target detection with exceptional efficiency and accuracy requires the advancement of immunoassays. Optical immunoassays have demonstrated significant potential in clinical diagnosis, food safety, environmental protection, and other fields. Through the innovative and feasible combination of enzyme catalysis and optical immunoassays, notable progress has been made in enhancing analytical performances. Among the kinds of reporter enzymes, alkaline phosphatase (ALP) stands out due to its high catalytic activity, elevated turnover number, and broad substrate specificity, rendering it an excellent candidate for the development of various immunoassays. This review provides a systematic evaluation of the advancements in optical immunoassays by employing ALP as the signal label, encompassing fluorescence, colorimetry, chemiluminescence, and surface-enhanced Raman scattering. Particular emphasis is placed on the fundamental signal amplification strategies employed in ALP-linked immunoassays. Furthermore, this work briefly discusses the proposed solutions and challenges that need to be addressed to further enhance the performances of ALP-linked immunoassays.

Progress in Electrochemical Immunosensors with Alkaline Phosphatase as the Signal Label.

Electrochemical immunosensors have shown great potential in clinical diagnosis, food safety, environmental protection, and other fields. The feasible and innovative combination of enzyme catalysis and other signal-amplified elements has yielded exciting progress in the development of electrochemical immunosensors. Alkaline phosphatase (ALP) is one of the most popularly used enzyme reporters in bioassays. It has been widely utilized to design electrochemical immunosensors owing to its significant advantages (e.g., high catalytic activity, high turnover number, and excellent substrate specificity). In this work, we summarized the achievements of electrochemical immunosensors with ALP as the signal reporter. We mainly focused on detection principles and signal amplification strategies and briefly discussed the challenges regarding how to further improve the performance of ALP-based immunoassays.

Mollugin activates GLP-1R to improve cognitive dysfunction in type 2 diabetic mice.

AIMS: The incidence of diabetic cognitive dysfunction is increasing year by year, and it has gradually become a research hot spot. Studies have shown that glucagon-like peptide-1 receptor (GLP-1R) agonists can improve cognitive dysfunction in diabetic patients. This study focuses on whether small molecule GLP-1R agonists from traditional Chinese medicine (TCM) can improve the diabetic cognitive dysfunction. MATERIALS AND METHODS: The small molecules from TCM were screened by cell membrane chromatography (CMC) with GLP-1R-HEK293 cell membrane column. MTT assay, flow cytometry, immunofluorescence cytochemistry and other methods were used to determine the effects of mollugin on the apoptosis rate and reactive oxygen species (ROS) level of high glucose (HG)/hydrogen peroxide (H2O2) induced PC12 cells. Real-Time PCR was used to detect mRNA expression in mouse cerebral cortex. Water maze test was further used to confirm the effect of mollugin on cognitive dysfunction in T2DM mice. KEY FINDINGS: Mollugin bound to GLP-1R, promoted Ca2+ influx, increased insulin secretion and cAMP content in ß-TC-6 cells. Mollugin enhanced the cell viability, ameliorated apoptosis, reduced intracellular ROS levels in HG/H2O2-injured PC12 cells. Mollugin reduced the T2DM mice's escape latency, improved neuronal cell damage, decreased the expression of Pik3ca, Akt1 and Mapk1 mRNA in the cerebral cortex tissue. SIGNIFICANCE: The results suggest that mollugin could improve cognitive dysfunction in T2DM mice through activating GLP-1R/cAMP/PKA signal pathway.

Schisandrin B, a potential GLP-1R agonist, exerts anti-diabetic effects by stimulating insulin secretion.

Diabetes mellitus is a metabolic disease that is characterized by elevated blood sugar. Although glucagon-like peptide-1 receptor agonists (GLP-1RA) lower blood glucose in a glucose-dependent manner, most of them are macromolecule polypeptides. Macromolecular peptides are relatively expensive and inconvenient compared with small molecules. Therefore, this study sought to identify the small molecules binding to GLP-1R via cell membrane chromatography (CMC), confirm their agonistic activity, and further study its beneficial effects in a mouse model of type 2 diabetes mellitus (T2DM) induced by a combination of high-fat diet and streptozotocin. We used CMC, calcium imaging and molecular docking techniques to screen and identify the potential small molecule Schisandrin B (Sch B), which exhibits a strong binding effect to GLP-1R, from the small molecule library of traditional Chinese medicine. Through in-vitro experiments, we found that Sch B stimulated insulin secretion in ß-TC-6 cells, while GLP-1R antagonist Exendin9-39, adenylate cyclase inhibitor SQ22536, and protein kinase A (PKA) inhibitor H89 could significantly inhibit the insulin secretion induced by Sch B. In vivo, Sch B significantly improved fasting blood glucose levels, intraperitoneal glucose tolerance test damage, and the status of pancreatic tissue damage, and reduced serum insulin levels, total cholesterol, triglyceride and low density lipoprotein in T2DM mice. These results indicate that Sch B alleviates T2DM by promoting insulin release through the GLP-1R/cAMP/PKA signaling pathway, suggesting that Sch B may be a potential GLP-1RA, which is expected to provide a new therapeutic strategy for the prevention and treatment of T2DM.

Background-Quenched Aggregation-Induced Emission through Electrostatic Interactions for the Detection of Poly(ADP-ribose) Polymerase-1 Activity.

Poly(ADP-ribose) polymerase-1 (PARP1) is a potential biomarker and therapeutic target for cancers that can catalyze the poly-ADP-ribosylation of nicotinamide adenine dinucleotide (NAD+) onto the acceptor proteins to form long poly(ADP-ribose) (PAR) polymers. Through integration with aggregation-induced emission (AIE), a background-quenched strategy for the detection of PARP1 activity was designed. In the absence of PARP1, the background signal caused by the electrostatic interactions between quencher-labeled PARP1-specitic DNA and tetraphenylethene-substituted pyridinium salt (TPE-Py, a positively charged AIE fluorogen) was low due to the fluorescence resonance energy transfer effect. After poly-ADP-ribosylation, the TPE-Py fluorogens were recruited by the negatively charged PAR polymers to form larger aggregates through electrostatic interactions, thus enhancing the emission. The detection limit of this method for PARP1 detection was found to be 0.006 U with a linear range of 0.01~2 U. The strategy was used to evaluate the inhibition efficiency of inhibitors and the activity of PARP1 in breast cancer cells with satisfactory results, thus showing great potential for clinical diagnostic and therapeutic monitoring.

Erythropoietin ameliorates cognitive dysfunction in mice with type 2 diabetes mellitus via inhibiting iron overload and ferroptosis.

Type 2 diabetes mellitus (T2DM) is strongly associated with an increased risk of developing cognitive dysfunction. Numerous studies have indicated that erythropoietin (EPO) has neurotrophic effects. Ferroptosis has been reported to be associated with diabetic cognitive dysfunction. However, the impact of EPO on T2DM-associated cognitive dysfunction and its protective mechanism remain unclear. To evaluate the effects of EPO on diabetes-associated cognitive dysfunction, we constructed a T2DM mouse model and found that EPO not only decreased fasting blood glucose but also ameliorated hippocampal damage in the brain. The Morris water maze test indicated that EPO improved cognitive impairments in diabetic mice. Moreover, a ferroptosis inhibitor improved cognitive dysfunction in mice with T2DM in vivo. Furthermore, a ferroptosis inhibitor, but not other cell death inhibitors, mostly rescued high-glucose damaged PC12 cell viability. EPO had a similar effect as the ferroptosis inhibitor, which increased cell viability in the presence of a ferroptosis inducer. In addition, EPO reduced lipid peroxidation, iron levels, and regulated ferroptosis-related expression of proteins in vivo and in vitro. These findings indicate that EPO ameliorates T2DM-associated cognitive dysfunction, which might be related to decreasing iron overload and inhibiting ferroptosis.

Surface plasmon resonance detection of UV irradiation-induced DNA damage and photoenzymatic repair processes through specific interaction between consensus double-stranded DNA and p53 protein.

DNA damage, such as DNA lesions and strand breaks, impairs normal cell functions and failure in the DNA repair process could lead to gene mutation, cell apoptosis and disease occurrence. p53 is a tumor suppressor and DNA-binding protein, and DNA damage might affect their interaction and the subsequent p53 function. Herein, real-time monitoring of DNA damage and repair processes through DNA-p53 protein interaction was performed by surface plasmon resonance (SPR). The target DNA with consecutive pyrimidine nucleobases was first damaged upon UVC (254 nm) irradiation and then photoenzymatically repaired under UVA (365 nm) irradiation. The as-formed double-stranded (ds) DNA between probe DNA and normal, damaged or repaired target DNA was immobilized on the sensor chips, followed by the injection of p53 protein. By measuring the SPR signals under different cases, the DNA damage and repair processes could be conveniently monitored. The SPR signals were inversely proportional to the UVC doses ranging from 0.021 to 1.26 kJ m-2, providing a viable means for the quantification of the DNA damage level. The binding affinity between p53 and the dsDNA formed upon the hybridization of probe DNA and normal, damaged, or photoenzymatically repaired target DNA was estimated. This is the first report on measuring the equilibrium dissociation constant (KD) between the p53 protein and the dsDNA with photodamaged or repaired target sequences. The sensing strategy by SPR thus opens a new avenue for real-time measurement of the DNA damage and the repair processes.

Ultraviolet C Irradiation-Induced Dehybridization of Double-Stranded Oligonucleotides: Mechanism Investigation and Label-Free Measurement of the Photodamage Level.

Elucidating the mechanism and estimating the extent of conformation change of double-stranded DNA (dsDNA) upon ultraviolet (UV) exposure are of vital importance for understanding the DNA photodamage process. The existing research was mainly focused on the lesions of single-stranded DNA (ssDNA) and involved off-site measurement of the photodamage level. In this work, short-wavelength UV (UVC) (254 nm) irradiation was demonstrated to induce the dehybridization of dsDNA due to the loss of paring capacity of photodamaged pyrimidine nucleobases. The intrinsic programmability of dsDNA enabled researchers to rationally design the on-demand dehybridization sites. The spatial conformation switch of dsDNA caused by UVC irradiation could be evolved into a label-free sensing platform for the on-site measurement of the DNA photodamage level.

Biosensors for the Detection of Enzymes Based on Aggregation-Induced Emission.

Enzymes play a critical role in most complex biochemical processes. Some of them can be regarded as biomarkers for disease diagnosis. Taking advantage of aggregation-induced emission (AIE)-based biosensors, a series of fluorogens with AIE characteristics (AIEgens) have been designed and synthesized for the detection and imaging of enzymes. In this work, we summarized the advances in AIEgens-based probes and sensing platforms for the fluorescent detection of enzymes, including proteases, phosphatases, glycosidases, cholinesterases, telomerase and others. The AIEgens involve organic dyes and metal nanoclusters. This work provides valuable references for the design of novel AIE-based sensing platforms.

A High-Quality CdSe/CdS/ZnS Quantum-Dot-Based FRET Aptasensor for the Simultaneous Detection of Two Different Alzheimer's Disease Core Biomarkers.

The simultaneous detection of two different biomarkers for the point-of-care diagnosis of major diseases, such as Alzheimer's disease (AD), is greatly challenging. Due to the outstanding photoluminescence (PL) properties of quantum dots (QDs), a high-quality CdSe/CdS/ZnS QD-based fluorescence resonance energy transfer (FRET) aptasensor for simultaneously monitoring the amyloid-ß oligomers (AßO) and tau protein was proposed. By engineering the interior inorganic structure and inorganic−organic interface, water-soluble dual-color CdSe/CdS/ZnS QDs with a near-unity PL quantum yield (>90%) and mono-exponential PL decay dynamics were generated. The π−π stacking and hydrogen bond interaction between the aptamer-functionalized dual-color QDs and gold nanorods@polydopamine (Au NRs@PDA) nanoparticles resulted in significant fluorescence quenching of the QDs through FRET. Upon the incorporation of the AßO and tau protein, the fluorescence recovery of the QDs-DNA/Au NRs@PDA assembly was attained, providing the possibility of simultaneously assaying the two types of AD core biomarkers. The lower detection limits of 50 pM for AßO and 20 pM for the tau protein could be ascribed to the distinguishable and robust fluorescence of QDs and broad spectral absorption of Au NRs@PDA. The sensing strategy serves as a viable platform for the simultaneously monitoring of the core biomarkers for AD and other major diseases.

DNA walking system integrated with enzymatic cleavage reaction for sensitive surface plasmon resonance detection of miRNA.

Abnormal expression levels of miRNA are associated with various tumor diseases, for example, glioma tumors are characterized by the up-regulation of miRNA-182. Surface plasmon resonance (SPR) assay for miRNA-182 from glioma patients was performed via DNA walking amplification strategy. The duplex between aminated swing arm DNA (swDNA) and block DNA (blDNA), and aminated track DNA (trDNA) with a biotin tag were tethered on the poly(ethylene glycol) (PEG)-modified chips. Upon formation of miRNA/blDNA duplex, the SPR signal decreased with the walking process of swDNA, as the biotinylated fragment of trDNA (biotin-TTGGAGT) was detached from the sensor surface caused by the nicking endonuclease Nb.BbvCI. Such a repeated hybridization and cleavage cycle occurred continuously and the detachment of more biotinylated fragments of trDNA from the chips led to the attachment of fewer streptavidin (SA) molecules and then smaller SPR signals. MiRNA-182 with concentrations ranging from 5.0 fM to 1.0 pM could be readily determined and a detection limit of 0.62 fM was achieved. The proposed method was highly selective and possessed remarkable capability for evaluating the expression levels of miRNA-182 in serum samples from healthy donors and glioma patients. The sensing protocol holds great promise for early diagnosis of cancer patients.

Erythropoietin Mitigates Diabetic Nephropathy by Restoring PINK1/Parkin-Mediated Mitophagy.

Diabetic nephropathy (DN), one of the most detrimental microvascular complications of diabetes, is the leading cause of end-stage renal disease. The pathogenesis of DN is complicated, including hemodynamic changes, inflammatory response, oxidative stress, among others. Recently, many studies have demonstrated that mitophagy, especially PINK1/Parkin-mediated mitophagy, plays an important role in the pathogenesis of DN. Erythropoietin (EPO), a glycoprotein hormone mainly secreted by the kidney, regulates the production of erythrocytes. This research intends to explore the beneficial effects of EPO on DN and investigate related mechanisms. In in vitro experiments, we found that EPO promoted autophagic flux and alleviated mitochondrial dysfunction in terms of mitochondrial fragmentation, elevated mitochondrial ROS as well as the loss of mitochondrial potential, and lowered the apoptosis level in high-glucose-treated mesangial cells. Moreover, EPO increased protein expressions of PINK1 and Parkin, enhanced the co-localization of LC3 with mitochondria, Parkin with mitochondria as well as LC3 with Parkin, and increased the number of GFP-LC3 puncta, resulting in increased level of PINK1/Parkin-mediated mitophagy in mesangial cells. The knockdown of PINK1 abrogated the effect of EPO on mitophagy. In addition, in vivo experiments demonstrated that EPO attenuated renal injury, reduced oxidative stress, and promoted expressions of genes related to PINK1/Parkin-mediated mitophagy in the kidneys of DN mice. In summary, these results suggest that PINK1/Parkin-mediated mitophagy is involved in the development of DN and EPO mitigates DN by restoring PINK1/Parkin-mediated mitophagy.

Ratiometric electrochemical detection of miRNA based on DNA nanomachines and strand displacement reaction.

MicroRNAs (miRNAs) play an important role in regulating gene expression in cells. Abnormal expression of miRNAs has been associated with a variety of diseases. A ratiometric electrochemical method for miRNA detection based on DNA nanomachines and strand displacement reaction was developed. Signal probe with ferrocene label and reference probe with methylene blue label were immobilized on gold nanoparticle (AuNP)-coated magnetic microbeads (AuNP-MMBs). The miRNA triggers the strand displacement reaction and forms a duplex with the protect probe, releasing one end of the DNA walker (DW); the released DW hybridizes with the ferrocene (Fc)-labeled signal probe. The signal probe detached from AuNP-MMBs upon cleavage of the Nb.BbvCI enzyme. The oxidation peak of MB moieties on the reference probe remains unchanged and the signals of Fc moieties on the signal probe are inversely proportional to the concentrations of miRNA. The ratio between Fc moieties at 0.35 V and MB moieties at -0.22 V (vs. Ag/AgCl) was used to quantify the expression level of miRNA with a detection limit down to 0.12 fM. The ratiometric assay possesses a strong ability to eliminate interference from environmental changes, thus offering the high selectivity of miRNA from the complexed biosystems, holding great significance for miRNA sensing. A ratiometric assay with high selectivity of miRNA has been developed based on DNA nanomachines and strand displacement reaction.