Advancements in magnetic nanoparticle-based biosensors for point-of-care testing.

The significance of point-of-care testing (POCT) in early clinical diagnosis and personalized patient care is increasingly recognized as a crucial tool in reducing disease outbreaks and improving patient survival rates. Within the realm of POCT, biosensors utilizing magnetic nanoparticles (MNPs) have emerged as a subject of substantial interest. This review aims to provide a comprehensive evaluation of the current landscape of POCT, emphasizing its growing significance within clinical practice. Subsequently, the current status of the combination of MNPs in the Biological detection has been presented. Furthermore, it delves into the specific domain of MNP-based biosensors, assessing their potential impact on POCT. By combining existing research and spotlighting pivotal discoveries, this review enhances our comprehension of the advancements and promising prospects offered by MNP-based biosensors in the context of POCT. It seeks to facilitate informed decision-making among healthcare professionals and researchers while also promoting further exploration in this promising field of study.

Anti-oxidant effects of herbal residue from Shengxuebao mixture on heat-stressed New Zealand rabbits.

Heat stress can lead to hormonal imbalances, weakened immune system, increased metabolic pressure on the liver, and ultimately higher animal mortality rates. This not only seriously impairs the welfare status of animals, but also causes significant economic losses to the livestock industry. Due to its rich residual bioactive components and good safety characteristics, traditional Chinese medicine (TCM) residue is expected to become a high-quality feed additive with anti-oxidative stress alleviating function. This study focuses on the potential of Shengxuebao mixture herbal residue (SXBR) as an anti-heat stress feed additive. Through the UPLC (ultra performance liquid chromatography) technology, the average residue rate of main active ingredients from SXBR were found to be 25.39%. SXBR were then added into the basal diet of heat stressed New Zealand rabbits at the rates of 5% (SXBRl), 10% (SXBRm) and 20% (SXBRh). Heat stress significantly decreased the weight gain, as well as increased neck and ear temperature, drip loss in meat, inflammation and oxidative stress. Also, the hormone levels were disrupted, with a significant increase in serum levels of CA, COR and INS. After the consumption of SXBR in the basal diet for 3 weeks, the weight of New Zealand rabbits increased significantly, and the SXBRh group restored the redness value of the meat to a similar level as the control group. Furthermore, the serum levels T3 thyroid hormone in the SXBRh group and T4 thyroid hormone in the SXBRm group increased significantly, the SXBRh group showed a significant restoration in inflammation markers (IL-1ß, IL-6, and TNF-α) and oxidative stress markers (total antioxidant capacity, HSP-70, MDA, and ROS) levels. Moreover, the real-time fluorescence quantitative PCR analysis found that, the expression levels of antioxidant genes such as Nrf2, HO-1, NQO1, and GPX1 were significantly upregulated in the SXBRh group, and the expression level of the Keap1 gene was significantly downregulated. Additionally, the SXBRm group showed significant upregulation in the expression levels of HO-1 and NQO1 genes. Western blot experiments further confirmed the up-regulation of Nrf2, Ho-1 and NQO1 proteins. This study provides a strategy for the utilization of SXBR and is of great significance for the green recycling of the TCM residues, improving the development of animal husbandry and animal welfare.

A Chamber-Based Digital PCR Based on a Microfluidic Chip for the Absolute Quantification and Analysis of KRAS Mutation.

The Kirsten rat sarcoma virus gene (KRAS) is the most common tumor in human cancer, and KRAS plays an important role in the growth of tumor cells. Normal KRAS inhibits tumor cell growth. When mutated, it will continuously stimulate cell growth, resulting in tumor development. There are currently few drugs that target the KRAS gene. Here, we developed a microfluidic chip. The chip design uses parallel fluid channels combined with cylindrical chamber arrays to generate 20,000 cylindrical microchambers. The microfluidic chip designed by us can be used for the microsegmentation of KRAS gene samples. The thermal cycling required for the PCR stage is performed on a flat-panel instrument and detected using a four-color fluorescence system. "Glass-PDMS-glass" sandwich structure effectively reduces reagent volatilization; in addition, a valve is installed at the sample inlet and outlet on the upper layer of the chip to facilitate automatic control. The liquid separation performance of the chip was verified by an automated platform. Finally, using the constructed KRAS gene mutation detection system, it is verified that the chip has good application potential for digital polymerase chain reaction (dPCR). The experimental results show that the chip has a stable performance and can achieve a dynamic detection range of four orders of magnitude and a gene mutation detection of 0.2%. In addition, the four-color fluorescence detection system developed based on the chip can distinguish three different KRAS gene mutation types simultaneously on a single chip.

Relationship between serum HIF-1α and VEGF levels and prognosis in patients with acute cerebral infarction combined with cerebral-cardiac syndrome.

Objective: This research was conducted to discuss the recent prognosis of patients with acute cerebral infarction (ACI) combined with cerebral-cardiac syndrome (CCS). Method: Eighty-seven patients with ACI were selected, which were divided into the ACI group (52 patients) and the CCS group (35 patients) according to whether the CCS was combined, and another 30 health controls were selected as the control group. Serum hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) levels of subjects in each group at the 1st day, the 3rd day, and the 7th day after admission were measured by enzyme-linked immunosorbent assay. After discharge for 30 days, the National Institutes of Health Stroke Scale (NIHSS) and the modified Rankin Scale (mRS) score were utilized to evaluate the prognosis of patients. The role of serum HIF-1α and VEGF levels in the prognosis of ACI combined with CCS patients was assessed by receiver operating characteristic curve and the binary logistic regression analysis. Results: Higher serum HIF-1α and VEGF levels were observed in the CCS and ACI groups versus the control group, and the levels of which were even higher in the CCS group in comparison to the ACI group. According to the prognosis of the NIHSS score, fasting blood glucose (FBG), Acute Physiology and Chronic Health Evaluation II score, creatine kinase-MB (CK-MB), and HIF-1α and VEGF levels at the 7th day of admission were higher while Glasgow coma scale (GCS) score was lower in the poor prognosis group than those in the good prognosis group, and the area under the curve (AUC) of serum HIF-1α and VEGF levels was 0.895 (95% confident interval [CI], 0.786-1.000), and 0.855 (95% CI, 0.731-0.980). According to the prognosis of the mRS score, FBG, CK-MB, and HIF-1α and VEGF levels at the 7th day of admission were higher while GCS score was lower in the poor prognosis group than those in the good prognosis group, and the AUC of serum HIF-1α and VEGF levels was 0.850 (95% CI, 0.722-0.979) and 0.901 (95% CI, 0.798-1.000). The results of the binary logistic regression analysis revealed that HIF-1α and VEGF levels may be independent risk factors influencing the prognosis of ACI combined with CCS. Conclusion: Serum HIF-1α and VEGF have a good predictive value for assessing the recent prognosis of patients with ACI combined with CCS, which could be independent risk factors influencing the prognosis of disease.

Edaravone Dexborneol Alleviates Neuroinflammation by Reducing Neuroglial Cell Proliferation and Suppresses Neuronal Apoptosis/Autophagy in Vascular Dementia Rats.

More and more evidence shows that the pathological mechanism of vascular dementia (VD) is closely related to oxidative stress injury, cell apoptosis, autophagy, inflammatory response, excitatory amino acid toxicity, synaptic plasticity change, calcium overload, and other processes. Edaravone dexborneol (EDB) is a new type of neuroprotective agent that can improve the neurological damage caused by an ischemic stroke. Previous studies showed that EDB has effects on synergistic antioxidants and induces anti-apoptotic responses. However, it remains unclear whether EDB can affect apoptosis and autophagy by activating the PI3K/Akt/mTOR signaling pathway and its impact on the neuroglial cells. In this study, we established the VD model of rats by bilateral carotid artery occlusion to explore the neuroprotective effect of EDB and its mechanism. Morris Water Maze test was applied to assess the cognitive function of rats. H&E and TUNEL staining were applied to observe the cellular structure of the hippocampus. Immunofluorescence labeling was used to observe the proliferation of astrocytes and microglia. ELISA was applied to examine the levels of TNF-α, IL-1ß and IL-6, and RT-PCR was applied to examine their mRNA expression levels. Western blotting was applied to examine apoptosis-related proteins (Bax, Bcl-2, Caspase-3), autophagy-related proteins (Beclin-1, P62, LC3B), PI3K/Akt/mTOR signaling pathway proteins and their phosphorylation levels. The results indicated that EDB ameliorates learning and memory in rats subjected to the VD model, alleviates neuroinflammatory response by reducing the proliferation of the neuroglial cell and inhibits apoptosis and autophagy, which may be mediated by the PI3K/Akt/mTOR signaling pathway.

Enzyme-Assisted Nucleic Acid Amplification in Molecular Diagnosis: A Review.

Infectious diseases and tumors have become the biggest medical challenges in the 21st century. They are driven by multiple factors such as population growth, aging, climate change, genetic predispositions and more. Nucleic acid amplification technologies (NAATs) are used for rapid and accurate diagnostic testing, providing critical information in order to facilitate better follow-up treatment and prognosis. NAATs are widely used due their high sensitivity, specificity, rapid amplification and detection. It should be noted that different NAATs can be selected according to different environments and research fields; for example, isothermal amplification with a simple operation can be preferred in developing countries or resource-poor areas. In the field of translational medicine, CRISPR has shown great prospects. The core component of NAAT lies in the activity of different enzymes. As the most critical material of nucleic acid amplification, the key role of the enzyme is self-evident, playing the upmost important role in molecular diagnosis. In this review, several common enzymes used in NAATs are compared and described in detail. Furthermore, we summarize both the advances and common issues of NAATs in clinical application.

Naked-Eye Detection of Food-Borne Pathogens Using Multiplex Hyperbranched Rolling Circle Amplification and Magnetic Particles.

Food safety is a significant public health issue in both developed and developing countries. Previous detection methods struggle to meet the current demands. We have proposed a new way to detect pathogens, allowing detection to be visualized by the naked eye. Using our newly developed assay, when target genes are present in the reaction, corresponding padlock probes form closed-loop molecules. Each reaction tube contains a pair of universal primers for identifying target genes. The ring padlock probes and corresponding universal primers start hyperbranched rolling circle amplification (HRCA) under the action of the polymerase, so as to gain branched chain amplification products, which are irreversibly entangled with magnetic particles to form aggregated magnetic particle clusters, and the detection results are visible to naked eyes. On the contrary, by using linear probes, the clustering of magnetic particles will not be produced. This method was applied to the detection of five food-borne pathogens enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), enteroinvasive Escherichia coli (EIEC) and Escherichia coli (E. coli), with detection limits of 1 × 103, 1 × 104, 1 × 103, 1 × 104 and 1 × 102 CFU/mL, respectively. This method can realize multiplex automatic detection of nucleic acid and shows great development potential in the field of molecular diagnosis.

Immune repertoire analysis of normal Chinese donors at different ages.

OBJECTIVES: This study investigated the characteristics of the immune repertoire in normal Chinese individuals of different ages. MATERIALS AND METHODS: In this study, all seven receptor chains from both B and T cells in peripheral blood of 16 normal Chinese individuals from two age groups were analyzed using high-throughput sequencing and dimer-avoided multiplex PCR amplification. Normal in this study is defined as no chronic, infectious or autoimmune disease within 6 months prior to blood draw. RESULTS: We found that compared with the younger group, the clonal expression of T-cell receptor repertoire increased in the older group, while diversity decreased. In addition, we found that the T-cell receptor repertoire was more significantly affected by age than the B-cell receptor repertoire, including significant differences in the use of the unique TCR-alpha and TCR-beta V-J gene combinations, in the two groups of normal participants. We further analyzed the degree of complementarity determining region 3 sequence sharing between the two groups, and found shared TCR-alpha, TCR-gamma, immunoglobulin-kappa and immunoglobulin-lambda chain complementarity determining region 3 sequences in all subjects. CONCLUSION: Taken together, our study gives us a better understanding of the immune repertoire of different normal Chinese people, and these results can be applied to the treatment of age-related diseases. Immune repertoire analysis also allows us to observe participant's wellness, aiding in early-stage diagnosis.

Recent Advances of Human Leukocyte Antigen (HLA) Typing Technology Based on High-Throughput Sequencing.

The major histocompatibility complex (MHC) in humans is a genetic region consisting of cell surface proteins located on the short arm of chromosome 6. This is also known as the human leukocyte antigen (HLA) region. The HLA region consists of genes that exhibit complex genetic polymorphisms, and are extensively involved in immune responses. Each individual has a unique set of HLAs. Donor-recipient HLA allele matching is an important factor for organ transplantation. Therefore, an established rapid and accurate HLA typing technology is instrumental to preventing graft-verses-host disease (GVHD) in organ recipients. As of recent, high-throughput sequencing has allowed for an increase read length and higher accuracy and throughput, thus achieving complete and high-resolution full-length typing. With more advanced nanotechnology used in high-throughput sequencing, HLA typing is more widely used in third-generation single-molecule sequencing. This review article summarizes some of the most widely used sequencing typing platforms and evaluates the latest developments in HLA typing kits and their clinical applications.

Recent advances and application of whole genome amplification in molecular diagnosis and medicine.

Whole genome amplification (WGA) is a technology for non-selective amplification of the whole genome sequence, first appearing in 1992. Its primary purpose is to amplify and reflect the whole genome of trace tissues and single cells without sequence bias and to provide sufficient DNA template for subsequent multigene and multilocus analysis, along with comprehensive genome research. WGA provides a method to obtain a large amount of genetic information from a small amount of DNA and provides a valuable tool for preserving limited samples in molecular biology. WGA technology is especially suitable for forensic identification and genetic disease research, along with new technologies such as next-generation sequencing (NGS). In addition, WGA is also widely used in single-cell sequencing. Due to the small amount of DNA in a single cell, it is often unable to meet the amount of samples needed for sequencing, so WGA is generally used to achieve the amplification of trace samples. This paper reviews WGA methods based on different principles, summarizes both amplification principle and amplification quality, and discusses the application prospects and challenges of WGA technology in molecular diagnosis and medicine.

[Comparison of therapeutic efficacy of Wuling Capsules prepared with different methods for rats with syndrome of liver Qi stagnation, spleen deficiency, and blood stasis].

Wuling Capsules is one of the commonly used drugs for the clinical treatment of chronic hepatitis B with the syndrome of liver Qi stagnation, spleen deficiency, and blood stasis. However, the present preparation method of Wuling Capsules ignores some macromolecules like polysaccharides. In this study, the influences of different ethanol concentrations in the preparation process on the extraction rates of macro-and micro-molecules were investigated. Further, the therapeutic efficacy of Wuling Capsules was evaluated with the reserpine-induced rat model of liver Qi stagnation, spleen deficiency, and blood stasis. When 50% ethanol was used for the last time of extraction, the concentrations of polysaccharides, salvianolic acid B, and schisandrin in the extract, as well as the dry extract yield, increased significantly compared with those of the original preparation method. However, the fingerprints of micro-molecules showed little difference between the two methods, with a similarity of 0.862. The study then set the 50% ethanol extraction as the new preparation method. The pharmacodynamics evaluation showed that the Wuling Capsules prepared with the original and new methods both significantly alleviated the emotional depression and metabolic disturbance in model rats, demonstrating good performance in protecting the rats against gastric mucosal injuries, modulating intestinal function, and activating blood circulation. The mechanism of action may be related to the regulation of gastrointestinal hormone secretion, reduction of inflammation, and promotion of dopamine synthesis in cortex and hippocampus. At the same dose, the Wuling Capsules prepared with the original and new methods showed roughly the same overall therapeutic efficacy. However, the Wuling Capsules prepared with the new method had stronger effect in activating blood circulation and modulating inflammation, but weaker effects in regulating gastrin and dopamine. The present study provides basis data for optimizing the preparation process of Wuling Capsules and deciphering the mechanism of its therapeutic effect on liver Qi stagnation, spleen deficiency, and blood stasis.

A novel approach for modulating the spatial distribution of fat globules in acid milk gel and its effect on the perception of fat-related attributes.

Modulating the inhomogeneous distribution of fat globules within an emulsion gel is now being considered an effective method to increase the perception of fat-related sensory attributes. However, the methods for preparing the inhomogeneous gel were relatively complicated in previous studies. In the present study, milks enriched with different sizes of fat globule were obtained and then used to prepare glucono-δ-lactone-induced milk gels. The gels with different spatial distributions of fat globules were obtained through natural creaming. To ensure the high fat content layer exist on the gel surface, the two gels made from the same milk were superimposed from the bottom to form a new gel. In situ confocal microscopy showed that under the same overall fat content, the superimposed gel containing larger fat globules (L-L gel) exhibited the greatest inhomogeneity in microstructure with the highest average surface fat area fraction (10.9%), and the largest difference in fat content between the surface and the inside layers (9.1%). To illustrate the effect of inhomogeneous distribution of fat globules in gels on the perception of fat-related attributes, quantitative descriptive sensory analysis as well as the lubrication properties measurement under simulated oral processing conditions were carried out. The results showed the superimposed gels exhibited higher creaminess ratings and lower friction coefficients at 20 mm/s than those of the original gels. Overall, the study modulated the spatial distribution of fat globules in acid milk gels through natural creaming and superimposition and illustrated its positive effect on the perception of fat-related sensory attributes.

Characterization of the relationship between olfactory perception and the release of aroma compounds before and after simulated oral processing.

Aroma is an important property of fermented milk, and it directly affects consumer acceptance. However, previous studies have mainly focused on analyzing the composition of aroma compounds in fermented milk in vitro, and the composition may be different from the real aroma composition that stimulates the sense of smell. Furthermore, the relationship between olfactory attributes and the release of aroma compounds was not fully understood. In this study, we selected 6 samples of fermented milk differing in aroma perception intensity based on our pretest. A descriptive sensory analysis focusing on orthonasal and retronasal olfaction of fermented milk was first conducted by semitrained panelists. Artificial saliva was mixed with the fermented milk samples and continuously stirred at 37°C for 15 s to simulate oral processing conditions. Headspace solid-phase microextraction-gas chromatography coupled with quadrupole time-of-flight mass spectrometry was applied to identify the head space composition of 6 kinds of fermented milk before and after the simulated oral processing. Twenty-five volatile compounds were identified in the fermented milks, 15 of which were predicted to have an influence on the olfactory perception of fermented milks during oral processing. Partial least squares regression analysis based on chemical and sensory data was then applied to explore the correlation between sensory perception and volatile aroma release. The results showed that oral processing greatly increased the perception of creamy aroma compounds, such as diacetyl and acetone, but did not increase the perception of dairy sour aroma compounds, such as butanoic acid and hexanoic acid. This study can help improve our understanding of the relationship between olfactory perceptions and the release of volatile aroma compounds under oral processing. It might also contribute to the design of palatable fermented milks catering to specific consumer preferences.

Determination of Rare Earth Elements in Pore Water Samples of Marine Sediments Using an Offline Preconcentration Method.

The concentrations of dissolved yttrium and rare earth elements (REY) in sediment pore water provide important geochemical information. However, due to the low REY concentration, complex matrix, and limited sample volume (often only a few milliliters), analysis of the REY in pore water often is highly challenging. In this study, a method was established to determine the dissolved REY in pore water of marine sediments using an offline preconcentration step with the ethylenediaminetriacetate chelating resin, followed by inductively coupled plasma-mass spectrometry. In addition, using a commercially available automated trace-element preconcentration system, the preconcentration step can be fully automated, saving labor and providing a better control of the final elution volume. The experimental conditions (pH, elution volume, elution acid concentration, and organic complexation effect) were assessed, and the optimal conditions were chosen. In particular, the organic complexation effect was found to be negligible. The procedure blank and limit of detection were satisfactory for studying REY in pore water of marine sediments, and the method also yielded satisfactory recoveries for the REY elements (83-110%). The method was then applied to analyze the dissolved REY concentrations of pore water samples collected in a sediment core (~ 30 cm) in the central Indian Ocean. The vertical distribution, dissolved REY concentration, and the average Post Archean Australian Shale-normalized pattern of the REY showed similarities to the previously published pore water REY data. This method provides an accurate yet facile approach for the analysis of all 15 REY in marine pore water samples using the sample volume of only ~ 5 mL.

Advances in Aptamer Screening and Drug Delivery.

Chemotherapy is the most commonly used treatment for cancer. However, due to their short circulation time and lack of specific biological distribution, chemotherapeutic drugs cause severe systemic toxicity. Using the agents specifically binding to the targeted molecules might resolve this dilemma. Aptamers can directly connect with the drugs or couple with the nano-carrier to reduce systemic toxicity. In this review, we elucidated the definition, characteristics and screening process of aptamers. The methods of drug delivery by aptamers were illustrated in details. Furthermore, clinical application of aptamers in recent years was briefly summarized and its long-term prospects were put forward.

Precise discrimination of Luminal A breast cancer subtype using an aptamer in vitro and in vivo.

Precise discrimination of breast cancer remains a challenge in clinical medicine, which depends on the development of novel specific molecular probes. However, the current technologies and antibodies cannot achieve precise discrimination of breast cancer subtypes very well. To address this problem, a novel truncated DNA aptamer MF3Ec was developed in this work. Aptamer MF3Ec exhibited high specificity and binding affinity against MCF-7 breast cancer cells with a Kd value of 18.95 ± 2.9 nM which is four times lower than that of the original aptamer, and could work at 4 °C, 25 °C and 37 °C with no obvious differences. Besides, aptamer MF3Ec displayed better stability in serum samples with a long existence time of about 12 h. Moreover, fluorescence imaging experiments indicated that aptamer MF3Ec was able to distinguish MCF-7 breast cancer cells from SK-BR-3, MDA-MB-231 and MCF-10A breast cancer cell subtypes, and differentiate the tumor-bearing mice and xenografted tissue sections of MCF-7 breast cancer cells from those of MDA-MB-231 and SK-BR-3 breast cancer cells in vivo and in vitro, respectively. Finally, clinical experiments indicated that aptamer MF3Ec could distinguish Luminal A breast cancer subtype from Luminal B (HER2+), HER2-enriched, and triple-negative breast cancer subtypes, para-carcinoma tissues and normal breast tissues. Collectively, all these results suggest that aptamer MF3Ec is a promising probe for precise discrimination and targeted therapy of Luminal A breast cancer molecular subtype.

[Study on rapid quality analysis method of Shengxuebao Mixture].

A rapid analysis method based on ultraviolet-visual(UV-Vis) spectroscopy, near infrared(NIR) spectroscopy and multivariable data analysis was established for quality evaluation of Shengxuebao Mixture. The contents of eight active ingredients of Shengxuebao Mixture including albiflorin, paeoniflorin, 2, 3, 5, 4'-tetra-hydroxy-stilbene-2-O-ß-D-glucopyranoside, specnuezhenide,ecliptasaponin D, emodin, calycosin-7-glucoside and astragaloside Ⅳ were simultaneously detected by using this method. HPLC-UV-MS was used as a reference method for determining the contents of these ingredients. Partial least squares(PLS) analysis was implemented as a linear method for multivariate models calibrated between UV spectrum/NIR spectrum and contents of 8 ingredients. Finally, the performance of the model was evaluated by 24 batches of test samples. The results showed that both UV-Vis and NIR models gave a good calibration ability with an R~2 value above 0.9, and the prediction ability was also satisfactory, with an R~2 value higher than 0.83 for UV-Vis model and higher than 0.79 for NIR model. The overall results demonstrate that the established method is accurate, robust and fast, therefore, it can be used for rapid quality evaluation of Shengxuebao Mixture.

Application of magnetic nanoparticles in nucleic acid detection.

Nucleic acid is the main material for storing, copying, and transmitting genetic information. Gene sequencing is of great significance in DNA damage research, gene therapy, mutation analysis, bacterial infection, drug development, and clinical diagnosis. Gene detection has a wide range of applications, such as environmental, biomedical, pharmaceutical, agriculture and forensic medicine to name a few. Compared with Sanger sequencing, high-throughput sequencing technology has the advantages of larger output, high resolution, and low cost which greatly promotes the application of sequencing technology in life science research. Magnetic nanoparticles, as an important part of nanomaterials, have been widely used in various applications because of their good dispersion, high surface area, low cost, easy separation in buffer systems and signal detection. Based on the above, the application of magnetic nanoparticles in nucleic acid detection was reviewed.

A simple fluorescence aptasensor for gastric cancer exosome detection based on branched rolling circle amplification.

Exosomes are membrane nanovesicles carrying molecular information that may reflect the biological and genetic characteristics of their parent cells. Numerous studies have demonstrated the potential of exosomes as noninvasive cancer biomarkers. Hence, specific detection of cancer cell-derived exosomes is of significant importance. Here, we developed a fluorescence assay for the determination of gastric cancer exosomes based on branched rolling circle amplification (BRCA) and an aptamer to target specific exosomes. The designed padlock probe was cyclized after incubation with an aptamer binding with the target exosome. BRCA was triggered by adding a second primer and the resulting long tandem double-stranded DNA product was detected using SYBR Green I as the fluorescent dye. This method demonstrated a high specificity for target exosomes with a detection limit of 4.27 × 104 exosomes per mL. Moreover, plasma from gastric cancer patients was tested to verify the clinical applicability of this assay. Our results demonstrated that this aptamer-based biosensor may show potential for the early diagnosis of gastric cancer.

Development of the iCubate Molecular Diagnostic Platform Utilizing Amplicon Rescue Multiplex Polymerase Chain Reaction.

We utilized Amplicon-Rescue Multiplex PCR (ARM-PCR) and microarray hybridization to develop and validate the iC-GPC Assay, a multiplexed, in vitro diagnostic test that identifies five of the most common gram positive bacteria and three clinically relevant resistance markers associated with bloodstream infections (BSI). The iC-GPC Assay is designed for use with the iC-System™, which automates sample preparation, ARM-PCR, and microarray detection within a closed cassette. Herein, we determined the limit of detection for each of the iC-GPC Assay targets to be between 3.0 × 105-1.7 × 107 CFU/mL, well below clinically relevant bacterial levels for positive blood cultures. Additionally, we tested 106 strains for assay inclusivity and observed a target performance of 99.4%. 95 of 96 non-target organisms tested negative for cross-reactivity, thereby assuring a high level of assay specificity. Overall performance above 99% was observed for iC-GPC Assay reproducibility studies across multiple sites, operators and cassette lots. In conclusion, the iC-GPC Assay is capable of accurately and rapidly identifying bacterial species and resistance determinants present in blood cultures containing gram positive bacteria. Utilizing molecular diagnostics like the iC-GPC Assay will decrease time to treatment, healthcare costs, and BSI-related mortality.