Magnetic Graphene Oxide Nanocomposites for Selective miRNA Separation and Recovery.

In this study, we developed magnetic graphene oxide composites by chemically attaching Fe3O4 nanoparticles to graphene oxide nanosheets. Characterization techniques, including Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), Raman spectroscopy, thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and transmission electron microscopy (TEM), confirmed the successful synthesis of Fe3O4@GO composites with desirable properties. The resulting composites exhibited superparamagnetic behavior, solubility, and compatibility for efficient miRNA separation. Using miR-29a as a model, we demonstrated the effective binding of miR-29a to the magnetic graphene oxide (GO) composites at an optimal concentration of 1.5 mg/mL, followed by a simple separation using magnetic forces. Additionally, the addition of 5.0 M urea enhanced the miRNA recovery. These findings highlight the potential use of our magnetic graphene oxide composites for the efficient separation and recovery of miR-29a, suggesting their broad applicability in various miRNA-based studies. Further exploration can focus on investigating endogenous miRNAs with aberrant expression patterns, contributing to the advancements in precision medicine.

Development of a High-Accuracy, Low-Cost, and Portable Fluorometer with Smartphone Application for the Detection of Urinary Albumin towards the Early Screening of Chronic Kidney and Renal Diseases.

This study presents the development of a portable fluorometer with a smartphone application designed to facilitate the early screening of chronic kidney and renal diseases by enabling the sensitive detection of urinary albumin. Utilizing a fluorescence-based aptasensor, the device achieved a linear calibration curve (0.001-1.5 mg/mL) with a linearity of up to 0.98022 and a detection limit of 0.203 µg/mL for human serum albumin (HSA). The analysis of 130 urine samples demonstrated comparable performance between this study's fluorometer, a commercial fluorometer, and the standard automated method. These findings validate the feasibility of the portable fluorometer and aptasensor combination as a reliable instrument for the sensitive and specific measurement of HSA in urine samples. Moreover, the fluorometer's portability offers potential applications in portable point-of-care testing, enhancing its utility in clinical settings for early disease screening.

Isothermal Amplification Technology for Disease Diagnosis.

Isothermal amplification (IA) is a nucleic acid amplification technology (NAAT) that has contributed significantly to the healthcare system. The combination of NAAT with a suitable detection platform resulted in higher sensitivity, specificity, and rapid disease diagnosis. Traditional NAAT, such as polymerase chain reaction (PCR), is widely applied in the general healthcare system but is rarely accessed in resource-limited hospitals. Some IA methods provide a rapid, sensitive, specific, and simple method for disease diagnosis. However, not all IA techniques have been regularly used in clinical applications because different biomarkers and sample types affect either the enzyme in the IA system or sample preparation. This review focuses on the application of some IA techniques that have been applied in the medical field and have the potential for use at points of care.

Detection of a miRNA biomarker for cancer diagnosis using SERS tags and magnetic separation.

Detection of miR-29a, a biomarker of cancers, using SERS tags and magnetic separation is described. The assay was designed to detect the miR-29a sequence by taking the complementary sequence and splitting it into a capture and detection probe. The SERS tags comprised the highly Raman active molecule 4-mercaptobenzoic acid (4-MBA) and DNA detection probes assembled onto the surface of gold nanorods (AuNRs) through the self-assembly process. The capture DNA conjugated magnetic nanoparticles (MNPs) were applied as capture probes. The detection was based on the hybridisation and sandwich complex formation. The resultant hybridisation-dependent complexes were recovered and enriched from the samples by magnetic separation. The enriched solution containing target miRNA hybridised with capture probes were dropped on a foil-covered slide to form a droplet for SERS analysis. A characteristic spectrum of 4-MBA was observed to indicate the presence of the miR-29a in the samples. The sensitivity of the assay is examined by measuring the SERS signal of the samples containing different concentrations of the miR-29a. The SERS intensity appears to increase with the concentration of miR-29a. The limit of detection (LOD) was found to be 10 pM without any amplification process. In addition, the selectivity and feasibility of the assay in complex media are evaluated with the non-target miRNAs comprising different sequences from the target miR-29a. The system was capable of detecting the target miR-29a specifically with high selectivity. These results suggest that this solution-based SERS platform has a significant capability for simple, sensitive, and selective miR-29a analysis.

Albuminuria detection using graphene oxide-mediated fluorescence quenching aptasensor.

A simple and sensitive graphene oxide-mediated fluorescence quenching aptasensor is developed to quantify albuminuria in urine samples. The developed aptasensor used the specific target binding property of aptamer and fluorescence quenching property of graphene oxide to determine the concentration of human serum albumin in urine. The limit of detection of the developed platform is 0.05 µg.mL-1 and the detection range is 0.1-600 µg.mL-1, which covers the albuminuria concentration range present in normal human urine and the urine of the patient with chronic kidney disease. This approach can be modified to measure albuminuria using a high-throughput quantification platform and portable point of care testing. In addition, the production cost for one reaction is cheaper than those for the standard automated method. Therefore, this aptasensor has significant potential for commercialization and public use.•Our protocol is customized by using the fluorescence quenching property of graphene oxide and specific binding property of human serum albumin aptamer to detect human serum albumin in urine sample•The limit of detection of our developed platform is 0.05 µg.mL-1•The detection range of our aptasensor is 0.1-600 µg.mL-1.

Sensitive detection of albuminuria by graphene oxide-mediated fluorescence quenching aptasensor.

Albuminuria is a pathological condition wherein the human serum albumin (HSA) protein is present in abnormally excess amounts in the urine. A simple and sensitive graphene oxide-mediated fluorescence quenching aptasensor is developed to quantify albumin in urine samples and HSA in serum samples. The aptamer-bound HSA used in this aptasensor has hairpin structures, which are characteristic of the aptamer binding site. The limit of detection of the developed platform is 0.05 µg·mL-1 and the detection range is 0.1-14.0 µg·mL-1, which covers the albuminuria concentration range present in normal human urine and the urine of the patient with kidney diseases. This approach can be modified to measure HSA using a high-throughput quantification platform and portable point of care testing. In addition, the production cost for one reaction is cheaper than those for other standard automated methods. Therefore, this aptasensor has significant potential for commercialization and wide-scale public use.

Ultrasensitive detection of lung cancer-associated miRNAs by multiple primer-mediated rolling circle amplification coupled with a graphene oxide fluorescence-based (MPRCA-GO) sensor.

MicroRNAs (miRNAs) play important roles in gene regulation and have been reported as biomarkers in cancer diagnosis. Herein, we develop an isothermal miRNA detection platform based on the highly efficient, multiple primer-mediated rolling circle amplification method coupled with a graphene oxide-based fluorescence (MPRCA-GO) assay, using lung cancer-associated miRNAs (miR-21 and miR-210) and a reference miRNA (miR-16) as model targets. The combination of the designed ssDNA probe and T4 RNA ligase (T4 Rnl2) used in the MPRCA-GO assay allowed for single-base mismatch discrimination. In addition, the superfluorescence quenching ability of GO allowed for rapid fluorescence detection. The developed platform had a limit of detection as low as 0.87 fM and could detect target miRNAs in cancer cell lines and human serums. Therefore, the MPRCA-GO sensor has the potential for single nucleotide polymorphism (SNP) analysis and applications in clinical diagnostics.

Rolling circle amplification and graphene-based sensor-on-a-chip for sensitive detection of serum circulating miRNAs.

In this study, we developed a simple multiplex miRNA detection platform based on rolling circle amplification and the fluorescence quenching property of reduced graphene oxide. The detection platform could be applied on a microfluidics chip with a mobile system controller to eliminate contamination and to facilitate potential use in remote areas. As a proof of concept, two fluorescence-labeled ssDNA tags were used for detection of miR-29a and miR-144*, two miRNAs that are highly expressed in the blood circulation of some patients with cancer or tuberculosis. The circular ssDNA probes in this study were designed to have an advantage over padlock probes as they can be prepared in advance. Our multiplex miRNA detection platform exhibited high sensitivity and selectivity, with a limit of detection of 0.05 pmol. In addition, our platform could detect target miRNAs from the total miRNA population extracted from human serum or a cancer cell line. These results indicated that our miRNA sensor has the potential to provide simple and high throughput miRNA analysis for disease diagnosis and prognosis.

Graphene based aptasensor for glycated albumin in diabetes mellitus diagnosis and monitoring.

We selected and modified DNA aptamers specifically bound glycated human serum albumin (GHSA), which is an intermediate marker for diabetes mellitus. Our aptamer truncation study indicated that the hairpin-loop structure with 23 nucleotides length containing triple G-C hairpins and 15-nucleotide loop, plays an important role in GHSA binding. Fluorescent quenching graphene oxide (GO) and Cy5-labeled G8 aptamer were used in this study to develop simple and sensitive graphene based aptasensor for GHSA detection. The limit of detection (LOD) of our aptasensor was 50 µg/mL, which was lower than other existing methods. In addition, with the nuclease resistance system, our GHSA detection platform could also be used in clinical samples. Importantly, our approach could significantly reveal the higher levels of GHSA concentrations in diabetes than normal serums. These indicate that our aptasensor has a potential for diagnosis and monitoring of diabetes mellitus.

In Vitro and In Vivo Response to Low-Modulus PMMA-Based Bone Cement.

The high stiffness of acrylic bone cements has been hypothesized to contribute to the increased number of fractures encountered after vertebroplasty, which has led to the development of low-modulus cements. However, there is no data available on the in vivo biocompatibility of any low-modulus cement. In this study, the in vitro cytotoxicity and in vivo biocompatibility of two types of low-modulus acrylic cements, one modified with castor oil and one with linoleic acid, were evaluated using human osteoblast-like cells and a rodent model, respectively. While the in vitro cytotoxicity appeared somewhat affected by the castor oil and linoleic acid additions, no difference could be found in the in vivo response to these cements in comparison to the base, commercially available cement, in terms of histology and flow cytometry analysis of the presence of immune cells. Furthermore, the in vivo radiopacity of the cements appeared unaltered. While these results are promising, the mechanical behavior of these cements in vivo remains to be investigated.