Simranjeet Singh Sekhon

Analytical bioconjugates, aptamers, enable specific quantitative detection of Listeria monocytogenes

As a major human pathogen in the Listeria genus, Listeria monocytogenes causes the bacterial disease listeriosis, which is a serious infection caused by eating food contaminated with the bacteria. We have developed an aptamer-based sandwich assay (ABSA) platform that demonstrates a promising potential for use in pathogen detection using aptamers as analytical bioconjugates. The whole-bacteria SELEX (WB-SELEX) strategy was adopted to generate aptamers with high affinity and specificity against live L. monocytogenes. Of the 35 aptamer candidates tested, LMCA2 and LMCA26 reacted to L. monocytogenes with high binding, and were consequently chosen as sensing probes. The ABSA platform can significantly enhance the sensitivity by employing a very specific aptamer pair for the sandwich complex. The ABSA platform exhibited a linear response over a wide concentration range of L. monocytogenes from 20 to 2×10(6) CFU per mL and was closely correlated with the following relationship: y=9533.3x+1542.3 (R(2)=0.99). Our proposed ABSA platform also provided excellent specificity for the tests to distinguish L. monocytogenes from other Listeria species and other bacterial genera (3 Listeria spp., 4 Salmonella spp., 2 Vibrio spp., 3 Escherichia coli and 3 Shigella spp.). Improvements in the sensitivity and specificity have not only facilitated the reliable detection of L. monocytogenes at extremely low concentrations, but also allowed for the development of a 96-well plate-based routine assay platform for multivalent diagnostics.

Aptamer-based Sandwich Assay and its Clinical Outlooks for Detecting Lipocalin-2 in Hepatocellular Carcinoma (HCC)

We validated a single-stranded, DNA aptamer-based, diagnostic method capable of detecting Lipocalin-2 (LCN2), a biomarker from clinically relevant hepatocellular carcinoma (HCC) patient serum, in the sandwich assay format. Nine aptamers (LCN2_apta1 to LCN2_apta9) for LCN2 were screened with SELEX processes, and a sandwich pair (LCN2_apta2 and LCN2_apta4) was finally chosen using surface plasmon resonance (SPR) and dot blotting analysis. The result of the proposed aptamer sandwich construction shows that LCN2 was sensitively detected in the concentration range of 2.5–500 ng mL−1 with a limit of detection of 0.6 ng mL−1. Quantitative measurement tests in HCC patients were run on straight serum and were compared with the performance of the conventional antibody-based ELISA kit. The aptamer sandwich assay demonstrated an excellent dynamic range for LCN2 at clinically relevant serum levels, covering sub-nanogram per mL concentrations. The new approach offers a simple and robust method for detecting serum biomarkers that have low and moderate abundance. It consists of functionalization, hybridization and signal read-out, and no dilution is required. The results of the study demonstrate the capability of the aptamer sandwich assay platform for diagnosing HCC and its potential applicability to the point-of-care testing (POCT) system.

Advances in pathogen-associated molecules detection using Aptamer based biosensors

Pathogens are all around us in the environment and although advances in medical science have been made to protect against infection by pathogens, through the use of antibiotics, vaccination and fungicide, yet pathogens continue to threaten human life. The fast pathogen detection still remains an unresolved issue since conventional identification and detection methods are complex, costly and require minimum two to three days for detection. Thus there is an urgent need for the development of robust, sensitive and portable diagnostic tools that can lead to accurate and rapid detection of a variety of pathogens. Aptamers are small DNA and RNA oligonucleotides that can bind with high affinity and selectivity to a large number of targets like small and macromolecules from organic, inorganic and biological origin. Aptasensors are biosensors where aptamers are used in place of biological ligands to sense targeted analytes. Oligonucleotide-(both DNA, RNA), peptide- and peptidenucleic acid (PNA)-based aptasensors offer high reproducibility against a wide variety of targets (proteins, peptides, drugs, metabolites, metal ions, cells etc.) and are emerging as the most suitable candidates for analytical methods that use a very small amount (nano to microlitre) of analytes. Herein, the recent developments of aptasensors for the detection of pathogens have been discussed briefly.

Acute and chronic toxicity assessment and the gene expression of Dhb , Vtg , Arnt , CYP4 , and CYP314 in Daphnia magna exposed to pharmaceuticals

The exposure of environment to pharmaceuticals disturbs the aquatic ecosystem. In this study,the effect of commonly used pharmaceutical compounds such as caffeine, ibuprofen, aspirin, and tetracycline has been investigated by the toxicity assay using Daphnia magna. The acute (48 h) and chronic (21 days) toxicity assays have been performed using Daphnia magna. The lethal concentrations of caffeine, ibuprofen, aspirin, and tetracycline with 50% mortality (LC50) have been determined as ∼445.3, 91.5, 310, and 90.8 mg/L, respectively. In the chronic toxicity test, low concentrations such as 1%, 2%, 10%, 20% LC50 of the pharmaceuticals were used to expose the organisms for 21 days. The effect of these pharmaceuticals on the molecular responses in the organisms has been studied from the gene expression level of five different biomarkers in Daphnia magna (Dhb, Vtg, Arnt, CYP4, and CYP314) after 48 h and 21 days exposure to the testing pharmaceuticals. The results show that five genes show different responsive patterns when Daphnia magna was under stressful conditions caused by caffeine, ibuprofen, aspirin, and tetracycline. During exposure to caffeine, only the response for the Dhb gene increased in the acute test, whereas all other genes, except Dhb gene, were down regulated in the chronic text. In contrast, in the acute and chronic toxicity test for ibuprofen, the responses for Arnt and CYP4 genes were reduced, respectively. Moreover, after 48 h exposure to aspirin, all other genes, except the Dhb gene, were down regulated. In the chronic test using tetracycline, five genes expression of D. magna were affected.

Detecting and Discriminating Shigella sonnei Using an Aptamer-Based Fluorescent Biosensor Platform

In this paper, a Whole-Bacteria SELEX (WB-SELEX) strategy was adopted to isolate specific aptamers against Shigella sonnei. Real-time PCR amplification and post-SELEX experiment revealed that the selected aptmers possessed a high binding affinity and specificity for S. sonnei. Of the 21 aptamers tested, the C(t) values of the SS-3 and SS-4 aptamers (Ct = 13.89 and Ct = 12.23, respectively) had the lowest value compared to other aptamer candidates. The SS-3 and SS-4 aptamers also displayed a binding affinity (KD) of 39.32 ± 5.02 nM and 15.89 ± 1.77 nM, respectively. An aptamer-based fluorescent biosensor assay was designed to detect and discriminate S. sonnei cells using a sandwich complex pair of SS-3 and SS-4. The detection of S. sonnei by the aptamer based fluorescent biosensor platform consisted of three elements: (1) 5’amine-SS-4 modification in a 96-well type microtiter plate surface (N-oxysuccinimide, NOS) as capture probes; (2) the incubation with S. sonnei and test microbes in functionalized 96 assay wells in parallel; (3) the readout of fluorescent activity using a Cy5-labeled SS-3 aptamer as the detector. Our platform showed a significant ability to detect and discriminate S. sonnei from other enteric species such as E. coli, Salmonella typhimurium and other Shigella species (S. flexneri, S. boydii). In this study, we demonstrated the feasibility of an aptamer sensor platform to detect S. sonnei in a variety of foods and pave the way for its use in diagnosing shigellosis through multiple, portable designs.

Toxicoproteomic approaches for analysis of microbial community inhabiting Asian dust particles

In this review, we provide an overview of the bio-analytical approaches for proteomic analysis of Asian dust storms. Asian dust has been known to travel with high-speed eastward winds and affects air quality over Japan and even the western coast of the U.S. during severe occurrences. Several reports have shown that Asian dust particles have a negative effect on a wide range of industries and human health activities. Here we give a short overview of Asian dust feature and its proteomic analysis approaches including 2-DE PAGE, metaproteomic analysis of large environmental samples, and automatic techniques for dust microbial analysis. Following this, we discuss the detection system of Asian dust particles that can be integrated with biosensor platform.

Proteomic analysis of Daphnia magna exposed to caffeine, ibuprofen, aspirin and tetracycline

The toxicity of pharmaceuticals thrown indiscriminately adversely affects the environment. In this study, the impact of caffeine, ibuprofen, aspirin and tetracycline has been examined through toxicity assay based proteomic analysis. Proteomic profile in Daphnia magna was analyzed by two-dimensional electrophoresis with pH in the range 3 to 10. The LC50 of caffeine, ibuprofen, aspirin and tetracycline determined as 445.3, 91.5, 310 and 90 mg/L, respectively, was used to expose to D. magna grown for 48 hour before isolating total proteins. The results show that the expression pattern of protein’s profile in D. magna gets changed due to the toxicity of caffeine, ibuprofen, aspirin and tetracycline. A large number of proteins expressed differently in D. magna in comparison to those exposed to various pharmaceuticals. The results showed that 18 up- and 1 down-regulation spot in relation to caffeine exposure; 24 up- and 6 down-regulation spots in relation to ibuprofen exposure; 3 up- and 4 down-regulation spots in relation to aspirin exposure and 10 up- and 3 down-regulation spots in relation to tetracycline exposure have been observed. Thus, the present systemic approach using proteomic analysis is very helpful in screening novel biomarkers for pharmaceuticals induced hazardous effects.

Color reduction of melanin by lysosomal and peroxisomal enzymes isolated from mammalian cells

Lysosomes and peroxisomes are organelles with many functions in all eukaryotic cells. Lysosomes contain hydrolytic enzymes (lysozyme) that degrade molecules, whereas peroxisomes contain enzymes such as catalase that convert hydrogen peroxide (H2O2) to water and oxygen and neutralize toxicity. In contrast, melanin is known as a helpful element to protect the skin against harmful ultraviolet rays. However, a high quantity of melanin leads to hyperpigmentation or skin cancer in human. New materials have already been discovered to inhibit tyrosinase in melanogenesis; however, melanin reduction does not suggest their preparation. In this study, we report that the color intensity because of melanin decreased by the cellular activation of lysosomes and peroxisomes. An increase in the superficial intensity of lysosome and peroxisome activities of HeLa cells was observed. In addition, a decrease in the amount of melanin has also been observed in mammalian cells without using any other chemical, showing that the process can work in vivo for treating melanin. Therefore, the results of this study indicate that the amount of melanin decreases by the lysosome and peroxisome activity after entering the cells, and functional organelles are effective in color reduction. This mechanism can be used in vivo for treating melanin.

Immobilization of para-nitrobenzyl esterase-CLEA on electrospun polymer nanofibers for potential use in the synthesis of cephalosporin-derived antibiotics

The para-nitrobenzyl esterase (PNBE) was successfully immobilized onto electrospun polystyrene/poly(styrene-co-maleic anhydride) (PS/PSMA) nanofibers as cross-linked enzyme aggregates (CLEA). The maximum amount of PNBE immobilized on the PSPSMA nanofibers was determined to be 56.4 mg/g of nanofibers. The variation of pH and temperature has been observed to affect the enzymatic activity of the free and immobilized PNBE-CLEA. The immobilized PNBE-CLEA has been observed to show improved stability as compared to the native one. PNBE-CLEA has been found to retain more than 63% activity after 60 consecutive reuses. Finally, the immobilized PNBECLEA presented a high decomposition efficiency of cephalexin tosylate PNB ester. The durability determination of PNBE-CLEA immobilized nanofibers showed 70% of de-esterification after 10 cycles. These results demonstrate that PNBE-CLEA immobilized nanofiber could be used as an economical and environment friendly system for the catalytic process of paranitrobenzyl esters in pharmaceutical synthesis.

Stress specific Escherichia coli biosensors based on gene promoters for toxicity monitoring

Target specific biosensor Escherichia coli strains have been engineered for studying the stress action. Four different recombinant E. coli biosensors were constructed by fusing one promoter each of ung, rseA, ibpA, and yggX gene in pET-21a::lacZ system, which contains very effective color-readout sensing system. The responses of four bacteria sensor strains, UNGLacZ, RSEALacZ, IBPALacZ, and YGGXLacZ, have been characterized under treatment with mitomycin C (MMC), phenol, ethanol and hydrogen peroxide (H2O2), respectively. UNGLacZ responded strongly only to MMC (DNA damaging chemical), whereas no response has been observed with phenol, which is a membrane damage chemical. However, RSEALacZ gave significant response with phenol. In addition, ethanol and hydrogen peroxide caused dose-dependent induction from IBPALacZ and YGGXLacZ, respectively. Thus, the results of the present study demonstrate that four bacterial strains show significant and different responses based on various stress defense mechanisms, which these genes originally possessed in E. coli. The four bacterial biosensors can be potentially used in environmental Point-of-Care Testing (ePOCT) leading to quick, easy and early detection of toxic contaminants locally as well as resulting in real-time measurements.