Gold Nanoparticle-Based Colorimetric Sensors: Properties and Application in Detection of Heavy Metals and Biological Molecules.

During the last decade, advances have been made in nanotechnology using nanomaterials, leading to improvements in their performance. Gold nanoparticles (AuNPs) have been widely used in the field of sensor analysis and are also combined with certain materials to obtain the desired characteristics. AuNPs are commonly used as colorimetric sensors in detection methods. In developing an ideal sensor, there are certain characteristics that must be met such as selectivity, sensitivity, accuracy, precision, and linearity, among others. Various methods for the synthesis of AuNPs and conjugation with other components have been carried out in order to obtain good characteristics for their application. AuNPs can be applied in the detection of both heavy metals and biological molecules. This review aimed at observing the role of AuNPs in its application. The synthesis of AuNPs for sensors will also be revealed, along with their characteristics suitable for this role. In the application method, the size and shape of the particles must be considered. AuNPs used in heavy metal detection have a particle size of around 15-50 nm; in the detection of biological molecules, the particle size of AuNPs used is 6-35 nm whereas in pharmaceutical compounds for cancer treatment and the detection of other drugs, the particle size used is 12-30 nm. The particle sizes did not correlate with the type of molecules regardless of whether it was a heavy metal, biological molecule, or pharmaceutical compound but depended on the properties of the molecule itself. In general, the best morphology for application in the detection process is a spherical shape to obtain good sensitivity and selectivity based on previous studies. Functionalization of AuNPs with conjugates/receptors can be carried out to increase the stability, sensitivity, selectivity, solubility, and plays a role in detecting biological compounds through conjugating AuNPs with biological molecules.

Gel Protein Extraction's Impact on Conformational Epitopes of Linear Non-Tagged MPT64 Protein.

The production and purification of recombinant proteins are crucial to acquiring pure MPT64 protein. Due to the fact that protein epitopes may undergo conformational changes during purification, this study, therefore, investigated an effective rapid purification method to produce highly intracellular pure MPT64 protein without causing conformational changes in the epitope under denaturing conditions. MPT64 was isolated from E. coli and electrophoresed using gel SDS-PAGE. Then, the desired protein bands were excised and purified with two methods: electroelution and passive elution. The isolated protein was identified via peptide mass fingerprinting using MALDI-TOF MS and reacted with IgG anti-MPT64, and the cross-reactivity of the isolated protein with IgY anti-MPT64 was confirmed using Western blot. The results show that both of these methods produced pure MPT64 protein, and the MPT64 protein was confirmed based on the MALDI-TOF MS results. Neither of these two methods resulted in epitope changes in the MPT64 protein so it could react specifically with both antibodies. The yield of MPT64 protein was higher with electroelution (2030 ± 41 µg/mL) than with passive elution (179.5 ± 7.5 µg/mL). Thus, it can be inferred that the electroelution method is a more effective method of purifying MPT64 protein and maintaining its epitope than the passive elution method.

Improving of pelB-Secreted MPT64 protein released by Escherichia coli BL21 (DE3) using Triton X-100 and Tween-80.

pelB has been known as a successful signal peptide to translocate the protein target extracellularly in the Escherichia coli system. However, in our previous study, the yield of MPT64 protein extracellular recovery was still low and plenty of this protein was remain trapped in cytoplasm and periplasm. Recently, nonionic surfactants were efficiently reported to secrete recombinant protein extracellularly. Nonetheless, it must be clarified whether the surfactant supplementation can improve the yield of MPT64 extracellular protein significantly without giving impact on the structure of isolated MPT64 protein and can minimized the cell lysis effect. MPT64 protein secretion was carried out by comparing the effects of surfactants Tween 80 and Triton × 100 at various concentrations. Triton × 100 was able to increase the extracellular MPT64 protein gain up to 3 times higher than Tween 80 and it was in line with the greater level ratio of cell leakage of Triton × 100 compared to that of Tween 80. Similarly, the viable cell of the cultures decreased dramatically. However, both surfactants did not interfere the structure of MPT64 protein. In conclusion, Triton × 100 can be chosen as the supporting surfactant to assist the act of peptide signal in improving the resulting of MPT64 extracellular protein.

Purity of maltose-binding protein - Recombinant streptavidin expressed in Escherichia coli BL21 (pD861-MBP: 327892).

Nearly 95% of streptavidin which is expressed in Escherichia coli found as an inclusion body. Protein expressed in an inclusion body form requires further steps for the folding process related to its purification. Whereas the purity level of the recombinant streptavidin is very crucial mainly for the specification test in diagnostic system. In this study, we designed synthetic gene of streptavidin to be fused with maltose-binding protein (MBP) gene to enhance its solubility when expressed in E. coli BL21 (pD861-MBP: 327892) and purified using amylose resin with gradient column buffer. Based on the SDS-PAGE characterization, the majority of recombinant streptavidin was found in soluble than that of insoluble form. Recombinant streptavidin was found at its suitable size at 56.6 kDa in the soluble protein fraction with a concentration of 537.42 mg/L. The purest fraction of streptavidin recombinant was obtained at the 58th fraction in a concentration of 0.86 mg/L with purity level of 98.77%. Compared to the initial crude protein extract, the level of purity is lower, 6.03%. In summary, the MBP purification method improves the purity level and enhances the solubility of the recombinant streptavidin.

Inhibition of bacillus spores germination by cinnamon bark, fingerroot, and moringa leaves extract.

This study was intended to discover the natural food preservatives by comparing the antibacterial effect of the ethanolic extract of cinnamon bark, finger root, and moringa leaves toward Bacillus cereus both the vegetative cells and spores. The antibacterial activities of the investigated extracts were assessed against cells using the agar diffusion method. Whereas the sporicidal test was performed by observing the colony growth, after various times of incubation (1, 3, and 5 h). The investigated extracts produced inhibition in a diameter ranging from 10.6 to 35.3 mm, and it can be classified that the extract of cinnamon bark was the most potent extract to inhibit the vegetative cells form, followed by fingerroot and the moringa leaves extract. Consistently, the ethanolic extract of cinnamon bark and fingerroot significantly yielded sporicidal activities higher than the moringa leaves extract. Both extracts exerted sporicidal activity within 1 h of contact time at the lowest test concentration of 5% w/v, whereas moringa leaves extract required a longer contact time (5 h) at higher concentration of 20% w/v. It can be concluded that cinnamon bark and fingerroot extract have great potential as effective food preservative candidates to inhibit the B. cereus growth than moringa leaves extract.

Comparative study of cefixime and tetracycline as an evaluation policy driven by the antibiotic resistance crisis in Indonesia.

Antibiotic resistance is a serious threat that occurs globally in the health sector due to increased consumption of inappropriate antibiotics. Guidelines for prescribing antibiotics for ARTIs have been issued in general practice to promote rational antibiotic prescribing. This study was conducted to compare the effectiveness of cefixime and tetracycline as a solution to improve monitoring of appropriate antibiotic use in the treatment of ARTIs. All stock isolates were rejuvenated first, and cultured on standard media and Kirby-Bauer disc diffusion method was used for susceptibility testing in accordance with the Clinical and Laboratory Standard Institute's (CLSI) recommendations. Identification of bacteria from a single isolate was carried out to determine which bacteria were resistant to cefixime and tetracycline. A total of 466 single isolates of bacteria were analyzed, which showed a percentage of resistance to cefixime 38.0%, and tetracycline 92.86%. Bacterial isolates were resistant to cefixime and tetracycilne was a genus of Haemophilus, Streptococcus, Corynebacterium, Staphylococcus, and bordetella. Cefixime compared to tetracycline was proven to be superior in terms of the effectiveness of ARIs treatment.

Comparison of simple and rapid extracting methods of free-tags Mycobacterium tuberculosis protein 64 Recombinant Protein from polyacrylamide gel: Electroelution and the optimized passive elution.

In this study, the Mycobacterium tuberculosis protein 64 (MPT64) protein was constructed without any tags to facilitate the purification using column affinity chromatography, but the MPT64 must be obtained as a pure protein. This study was purpose to ensure the efficient extracting method to purify protein MPT64 directly from the polyacrylamide gel. The crude extract of extracellular protein containing MPT64 protein was separated into single protein band and the targeted protein which is located in the size of 24 kDa was excised. Each of the six bands was collected in a sterile microtube to be eluted using electroelution and the optimized of the passive-elution method. Both the elution methods demonstrated the purity level of the MPT64 protein by detecting a solely band on the gel at the 24 kDa. Among the variety of passive-elution time, the highest MPT64 protein concentration was 0.549 mg/ml after elution for 72 h. However, the electroelution result provided higher MPT64 protein concentration, i.e., 0.683 mg/mL. However, based on the recognition of the purified MPT64 protein on commercial detection kit of MPT64 protein, it showed that the positive result was only showed by the passive-elution extracting protein. Therefore, for purifying the protein MPT64 from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, the efficient method was passive elution.

Effect of water reservoirs types on the prevalence and antibiotic resistance profiles of Pseudomonas aeruginosa isolated from bathroom water in hospitals.

This study was aimed to isolate and characterize Pseudomonas aeruginosa antibiotic resistance profiles that isolated from bathroom water of five hospitals in Bandung, Indonesia, with different types of water reservoirs. Total of 25 water samples from bathrooms of five hospitals were collected and analyzed for the existence of P. aeruginosa colonies on the surface of MacConkey agar media using a streak plate method and identified using phenotypic identification and a series of biochemical tests. All P. aeruginosa isolates were tested against ceftazidime, piperacillin/tazobactam, ciprofloxacin, meropenem, and gentamicin containing in paper disc, using the agar diffusion method. Of all samples, the total number of P. aeruginosa isolates was less than that of non-P. aeruginosa. In hospitals that use permanent bathtubs, a greater total bacterial count was obtained than those using pails. From 110 isolates, 14.54% were multidrug resistance antibiotics. The majority of the resistant isolates were from hospital B with permanent bathtubs. Of 25 isolates from that hospital, P. aeruginosa isolates were resistant to ceftazidime (20%), piperacillin/tazobactam (4%), ciprofloxacin (20%), and gentamicin (20%). The multiple antibiotic resistance index value of P. aeruginosa isolates was 0.4-0.6. Thus, it can be concluded that the bathroom wáter in the hospital with permanent bathtubs were potential reservoirs of antibiotic-resistant P. aeruginosa.

Antibiotic resistance: Evaluation of levofloxacin treatment in acute respiratory tract infections cases at the Tasikmalaya City Health Center, Indonesia.

Acute respiratory tract infections (ARTIs) are an acute inflammation of the upper and lower respiratory tract caused by the infection of microorganisms or bacteria, viruses, without or accompanied by inflammation of the lung parenchyma. The use of antibiotics is one way to treat respiratory diseases. This study aims to determine the level of resistance of levofloxacin antibiotics to clinical isolates from ARTIs patients at the Tasikmalaya Health Center, Indonesia. The stages of the research included rejuvenation of clinical single isolates from ARTIs patients, identification of bacteria, and antibiotic resistance testing using the paper-disc method. The results of resistance tests from 142 single clinical isolates of acute respiratory infections showed that levofloxacin antibiotics had high levels of resistance of 50.0%, 30.95% of resistance with intermediate levels, and 19.04% were still sensitive. Bacterial identification test results showed bacteria that have been resistant to levofloxacin are from the genus Haemophillus, Streptococcus, Corynebacterium, Staphylococcus, and Bordetella. Treatment of ARTIs with the antibiotic levofloxacin shows that there has been a relatively large resistance, where the results of the identification of all bacteria showed the bacteria that cause ARTIs.

Real-time monitoring of rhamnose induction effect on the expression of mpt64 gene fused with pelB signal peptide in Escherichia coli BL21 (DE3).

In this research, Escherichia coli BL21 (DE3) harboring an expression vector constructed with a rhamnose-inducible promoter and a pelB signal peptide was used as a host cell to produce MPT64 protein. The objective of this research was to figure out the optimum time of mpt64 gene expression through real-time monitoring of MPT64 protein production and distribution in host compartments. The mpt64 expression was regulated by the rhamnose presence at a concentration of 4 mM. The real-time isolated protein was monitored using polyacrylamide gel electrophoresis in denaturation condition. Based on real-time monitoring, the MPT64 protein (24 kDa) in the cytoplasm was optimum detected at 24 h after induction. For periplasmic fraction, the protein was detected at 4 h after induction but thinning at 15 h after induction. At 16 h after induction, the MPT64 protein band was found in the medium with increasing concentrations until 24 h. Thus, it can be concluded that the mpt64 gene expression was regulated in the presence of rhamnose as an inducer, and the proteins were shown to be translocated throughout the host cell compartment with different levels of protein accumulation at different times, according to the role of pelB as a signal peptide.

Optimization of culture conditions for Mpt64 synthetic gene expression in Escherichia coli BL21 (DE3) using surface response methodology.

MPT64 is a specific protein that is secreted by Mycobacterium tuberculosis complex (MTBC). The objective of this study was to obtain optimum culture conditions for MPT64 synthetic gene expression in Escherichia coli BL21 (DE3) by response surface methodology (RSM). The RSM was undertaken to optimize the culture conditions under different cultivation conditions (medium concentration, induction time and inducer concentration), designed by the factorial Box-Bhenken using Minitab 17 statistical software. From the randomized combination, 15 treatments and three center point repetitions were obtained. Furthermore, expression methods were carried out in the flask scale fermentation in accordance with the predetermined design. Then, the MPT64 protein in the cytoplasm of E. coli cell was isolated and characterized using sodium dodecyl sulfate polyacrilamide electrophoresis (SDS-PAGE) then quantified using the ImageJ program. The optimum conditions were two-fold medium concentration (tryptone 20 mg/mL, yeast extract 10 mg/mL, and sodium chloride 20 mg/mL), 5 h of induction time and 4 mM rhamnose. The average concentration of recombinant MPT64 at optimum conditions was 0.0392 mg/mL, higher than the predicted concentration of 0.0311 mg/mL. In conclusion, the relationship between the selected optimization parameters strongly influenced the level of MPT64 gene expression in E. coli BL21 (DE3).

The ethanolic extract of ashitaba stem (Angelica keskei [Miq.] Koidz) as future antituberculosis.

Considering the easy contagion of tuberculosis (TB) disease spread and the emergence of multidrug-resistant TB, which directly impacts the failure of therapeutic goals and mortality rates increasing, TB disease control remains to be the main concern of continuous health development effort. Therefore, the discovery of new TB drug is needed. This research assessed the new natural anti-TB drug from the ethanolic extract of Angelica keiskei stem obtained from Lombok, Indonesia. The objectives of this study were to evaluate the sensitivity of Mycobacterium tuberculosis (Mtb) H37Rv strain to A. keiskei stem extract and to determine its minimum inhibitory concentration (MIC). The extraction methods of A. keiskei stem were done using a maceration method. In addition to phytochemical screening and water content analysis using standard method, the phytochemical parameters were analyzed by thin-layer chromatography. Ethanolic extract of A. keiskei stem was assayed for their Mtb inhibitory activity using the proportion method. The phytochemical analysis result showed that the secondary metabolites contain in the extract were flavonoid, polyphenol, tannin, monoterpenoid and sesquiterpen, quinon, and saponin. The anti-TB test result showed the active activity of ethanolic extract of A. keiskei against Mtb H37Rv strain with MIC ranging from 6% to 8% w/v. In conclusion, ethanolic extract of A. keiskei is a prospective natural anti-TB for the future.