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.

Effects of Boric Acid and Storage Temperature on the Analysis of Microalbumin Using Aptasensor-Based Fluorescent Detection.

The instability of human serum albumin (HSA) in urine samples makes fresh urine a requirement for microalbumin analyses using immunoturbidimetry. Here, we determined the ability of an aptasensor-based fluorescent platform to detect microalbumin in old, boric acid-preserved urine samples. Our results show that the cleavage site of protease enzymes on urine albumin protein differed from the binding position of the aptamer on HSA protein, suggesting the aptasensor may be effective for albumin detection in non-fresh urine. Furthermore, the addition of boric acid in urine samples over a short term (at ambient temperature (Ta) and 4 °C), long term (-20 and -80 °C), and following freeze-thawing (1-3 cycles) did not significantly affect albumin stability, as analyzed using the aptasensor. Therefore, boric acid stabilized has in urine stored over a short- and long-term. Thus, the aptasensor developed by us is applicable for HSA detection in boric acid-preserved urine that has been stored for 7-d at Ta and 4 °C, and in the long-term at -80 °C.

Dual sensitive and rapid detection of glycated human serum albumin using a versatile lead/graphene nanocomposite probe as a fluorescence-electrochemical aptasensor.

Monitoring of glycated human serum albumin (GHSA) as a glycemic marker for screening and monitoring of diabetes mellitus is widely practiced for patients with conditions that affect red blood cells. In this study, a complex comprising Pb ions adsorbed on graphene oxide (GO-Pb) was fabricated and utilized as a versatile probe in a fluorescence-electrochemical aptasensor for GHSA quantification. To simplify the aptasensor, the GO-Pb complex probe was prepared via an ion adsorption process. After modification with a fluorophore-labeled aptamer, the GO-Pb complex served as an excellent energy acceptor in fluorescence-based analysis, as well as generating a high current in the electrochemical transducer. Additionally, the proposed platform can detect GHSA via the dual technique from a single sample, allowing for precise and accurate results. Under optimal conditions, the fluorescence-electrochemical aptasensor exhibited a linear relationship with GHSA concentrations from 0.001 to 80 µg mL-1 and from 0.005 to 10 µg mL-1 for fluorescence and electrochemical detection, respectively. The corresponding detection limits were 8.80 ng mL-1 and 0.77 ng mL-1, respectively. The proposed aptasensor additionally displayed good selectivity and excellent stability. Moreover, its successful application in the analysis of clinical samples further demonstrated its utility. Therefore, the proposed platform has significant potential as a novel, facile, highly responsive, and low-cost monitoring method for the development of diabetes mellitus diagnostic devices intended for a clinical setting.

Immobilization-Free Electrochemical Sensor Coupled with a Graphene-Oxide-Based Aptasensor for Glycated Albumin Detection.

An immobilization-free electrochemical sensor coupled with a graphene oxide (GO)-based aptasensor was developed for glycated human serum albumin (GHSA) detection. The concentration of GHSA was monitored by measuring the electrochemical response of free GO and aptamer-bound GO in the presence of glycated albumin; their currents served as the analytical signals. The electrochemical aptasensor exhibited good performance with a base-10 logarithmic scale. The calibration curve was achieved in the range of 0.01-50 µg/mL. The limit of detection (LOD) was 8.70 ng/mL. The developed method was considered a one-drop measurement process because a fabrication step and the probe-immobilization process were not required. This simple sensor offers a cost-effective, rapid, and sensitive detection method, and could be an alternative approach for determination of GHSA levels.

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.

Lactococcus lactis KA-FF 1-4 reduces vancomycin-resistant enterococci and impacts the human gut microbiome.

Probiotic is an alternative method to treat intestinal infection disease caused by antibiotic-resistant bacteria. In this study, Lactococcus lactis KA-FF 1-4 demonstrated to have the potential to inhibit the growth of Vancomycin-resistant enterococci (VRE) by producing anti-microbial substance. In co-culture, L. lactis KA-FF 1-4 (108 CFU/mL) inhibited the growth of VRE from 103-104 CFU/mL to zero after 6 h of exposure. However, in a gut model contained human gut microbiota, this anti-VRE activity of L. lactis KA-FF 1-4 was reduced to only 3.59-6.12%. The unexpected difference in efficacy between the experimental models could be explained by the fact that the growth of L. lactis KA-FF 1-4 was stable in the gut model. Leaving aside these limitations, we observed that adding L. lactis KA-FF 1-4 into the human gut model containing VRE was able to enhance microbial richness and diversity. Specifically, a higher abundance of beneficial microbes from the group of Bifidobacterium spp. and Bacteroides fragilis. L. lactis KA-FF 1-4 also enhanced the abundance of Parabacteroides, Lactococcus, and Fusobacterium and promoted the production of lactic acid in the gut model. However, these effects were not observed in the gut model without L. lactis KA-FF 1-4. Even though this study could not demonstrate a significant anti-VRE effect of the L. lactis KA-FF 1-4 in a gut model, our results still offer evidence that L. lactis KA-FF 1-4 could positively modulate the gut microbiota by promoting the growth of beneficial microbes and their metabolite. L. lactis KA-FF 1-4 has probiotic properties to fight against VRE infection, therefore further investigation in animal model is needed.

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.

MULTILOCUS SEQUENCE TYPING OF BRUCELLA ISOLATES FROM THAILAND.

Although brucellosis outbreaks in Thailand are rare, they cause abortions and infertility in animals, resulting in significant economic loss. Because Brucella spp display > 90% DNA homology, multilocus sequence typing (MLST) was employed to categorize local Brucella isolates into sequence types (STs) and to determine their genetic relatedness. Brucella samples were isolated from vaginal secretion of cows and goats, and from blood cultures of infected individuals. Brucella species were determined by multiplex PCR of eight loci, in addition to MLST based on partial DNA sequences of nine house-keeping genes. MLST analysis of 36 isolates revealed 78 distinct novel allele types and 34 novel STs, while two isolates possessed the known ST8. Sequence alignments identified polymorphic sites in each allele, ranging from 2-6%, while overall genetic diversity was 3.6%. MLST analysis of the 36 Brucella isolates classified them into three species, namely, B. melitensis, B. abortus and B. suis, in agreement with multiplex PCR results. Genetic relatedness among ST members of B. melitensis and B. abortus determined by eBURST program revealed ST2 as founder of B. abortus isolates and ST8 the founder of B. melitensis isolates. ST 36, 41 and 50 of Thai Brucella isolates were identified as single locus variants of clonal cluster (CC) 8, while the majority of STs were diverse. The genetic diversity and relatedness identified using MLST revealed hitherto unexpected diversity among Thai Brucella isolates. Genetic classification of isolates could reveal the route of brucellosis transmission among humans and farm animals and also reveal their relationship with other isolates in the region and other parts of the world.