Bhagwan S. Batule

Ultrafast sonochemical synthesis of protein- inorganic nanoflowers

We developed a simple but efficient method to synthesize protein-inorganic hybrid nanostructures with a flower-like shape (nanoflowers), which relies on sonication to facilitate the synthesis of the nanoflowers. With this technique, we synthesized nanoflowers containing laccase as a model protein and copper phosphate within 5 minutes at room temperature. The resulting laccase nanoflowers yielded greatly enhanced activity, stability, and reusability, and their usefulness was successfully demonstrated by applying them in the colorimetric detection of epinephrine. The strategy developed could be used to rapidly synthesize nanoflowers for various applications in biosensor and enzyme catalysis and would expand the utilization of nanoflowers in diverse fields of biotechnology.

Rapid and ultrasensitive detection of microRNA by target-assisted isothermal exponential amplification coupled with poly (thymine)-templated fluorescent copper nanoparticles.

We devised a novel method for rapid and ultrasensitive detection of target microRNA (miRNA) by employing target-assisted isothermal exponential amplification (TAIEA) combined with poly (thymine)-templated fluorescent copper nanoparticles (CuNPs) as signaling probes. The target miRNA hybridizes to the unimolecular template DNA and works as a primer for the extension reaction to form double-stranded product, which consequently generates two nicking endonuclease recognition sites. By simultaneous nicking and displacement reactions, exponential amplification generates many poly (thymine) strands as final products, which are employed for the synthesis of fluorescent CuNPs. Based on the fluorescent signal from CuNPs, target miRNA is detected as low as 0.27 fM around 1 h of total analysis time. The diagnostic capability of this system has been successfully demonstrated by reliably detecting target miRNA from different cell lysates, showing its great potential towards real clinical applications.

A simple and eco-friendly one-pot synthesis of nuclease-resistant DNA–inorganic hybrid nanoflowers

A simple and eco-friendly method has been developed for the one-pot synthesis of DNA-copper nanoflowers that exhibit high loading efficiencies, low cytotoxicities, and strong resistance against nucleases.

Ultrasensitive DNA detection based on target-triggered rolling circle amplification and fluorescent poly(thymine)-templated copper nanoparticles

We describe a novel strategy for the ultrasensitive detection of target DNA based on rolling circle amplification (RCA) coupled with fluorescent poly(thymine)-templated copper nanoparticles (poly T-CuNPs). In the presence of target DNA, a padlock DNA probe that consists of two regions: a target DNA-specific region and a poly(adenine) region, is circularized by the ligation reaction, and the subsequent RCA reaction is promoted to generate long, concatemeric, single-stranded DNA (ssDNA) with a lot of repetitive poly T sequences. As a result, a large number of poly T-CuNPs are formed, exhibiting a highly fluorescent signal. However, in the absence of target DNA or in the presence of non-specific target DNA, the padlock DNA probe is not circularized and the subsequent RCA is not executed, leading to no production of fluorescent poly T-CuNPs. With this simple strategy, we successfully analyzed the target DNA with the ultralow detection limit of 7.79 aM, a value that is 3 or 7 orders of magnitude lower than those of previous RCA-based fluorescent DNA detection strategies. In addition, the developed system was demonstrated to selectively discriminate non-specific target DNAs with one-base mismatch, suggesting potential application in the accurate diagnosis of single nucleotide polymorphisms or mutations.

Barcode DNA-Mediated Signal Amplifying Strategy for Ultrasensitive Biomolecular Detection on Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

We have devised a barcode DNA-mediated signal amplifying strategy for ultrasensitive biomolecular detection by utilizing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). As a model target, thrombin was first captured by specific aptamer15 functionalized on magnetic beads (MBs-apt15) and sandwiched through the simultaneous interaction with gold nanoparticles modified with another specific aptamer29 and barcode DNA molecules (apt29-AuNPs-bcDNAs). The sandwiched complex was collected by convenient magnetic separation and then treated with potassium cyanide (KCN) to dissolve the gold nanoparticles (AuNPs) and consequently release the barcode DNA molecules (bcDNAs), which were then again magnetically separated and analyzed by using MALDI-TOF MS. Under optimized conditions, this strategy revealed an excellent sensitivity with a limit of detection of 0.89 aM in a wide linear detection range from 0 aM to 0.1 nM and exhibited an acceptable recovery for thrombin detection in complex biological matrices. This signal amplifying strategy based on MALDI-TOF MS could greatly enable the ultrasensitive detection of various low abundant biomolecules.

Colorimetric Detection of Norovirus in Oyster Samples through DNAzyme as a Signaling Probe

Worldwide, norovirus is one of the most associated causes of acute gastroenteritis, which leads to nearly 50 000 child deaths every year in developing countries. Therefore, there is great demand to develop a rapid, low-cost, and accurate detection assay for the foodborne norovirus infection to reduce mortality caused by norovirus. Considering the importance of norovirus, we have demonstrated a highly sensitive and specific colorimetric detection method for analysis of human norovirus genogroups I and II (HuNoV GI and GII) in oyster samples. This is the first report to employ colorimetric HRPzyme-integrated polymerase chain reaction (PCR) for direct norovirus detection from the real shellfish samples. We found that the HRPzyme-integrated PCR method is more sensitive than the gel electrophoresis approach and could detect the HuNoV GI and GII genome up to 1 copy/mL. The specificity of the proposed method was successfully demonstrated for HuNoV GI and GII. Further, we performed testing HuNoVs in the spiked oyster samples, and the HRPzyme-integrated PCR method proved to be an ultrasensitive and selective method for detecting HuNoVs in the real samples. By integration of the proposed method with the portable PCR machine, it would be more reliable to improve food safety by detecting HuNoVs in the different types of shellfish, such as oyster and mussel, at the production field.

Smartphone‐based portable wireless optical system for the detection of target analytes

Rapid and accurate on-site wireless measurement of hazardous molecules or biomarkers is one of the biggest challenges in nanobiotechnology. A novel smartphone-based Portable and Wireless Optical System (PAWS) for rapid, quantitative, and on-site analysis of target analytes is described. As a proof-of-concept, we employed gold nanoparticles (GNP) and an enzyme, horse radish peroxidase (HRP), to generate colorimetric signals in response to two model target molecules, melamine and hydrogen peroxide, respectively. The colorimetric signal produced by the presence of the target molecules is converted to an electrical signal by the inbuilt electronic circuit of the device. The converted electrical signal is then measured wirelessly via multimeter in the smartphone which processes the data and displays the results, including the concentration of analytes and its significance. This handheld device has great potential as a programmable and miniaturized platform to achieve rapid and on-site detection of various analytes in a point-of-care testing (POCT) manner.

Single-Step Recombinase Polymerase Amplification Assay Based on a Paper Chip for Simultaneous Detection of Multiple Foodborne Pathogens

A paper chip device-based recombinase polymerase amplification (RPA) method was developed for highly sensitive and selective single-step detection of foodborne pathogens. A paper chip was manufactured by simply stacking functional papers. RPA reagents and fluorescent probe were dried on the reaction zone of a patterned poly(ether sulfone) membrane. The RPA reaction was initiated by adding pathogen DNAs into an injection hole. Paper chip-based analysis of pathogens showed optimal performance at 37 °C for 20 min and the results were comparable to those obtained with solution-based RPA reactions. Based on the paper chip-based fluorescence signal, Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium were simultaneously detected with detection limits of 102 cfu/mL. The diagnostic utility of the device was demonstrated by the reliable detection of E. coli and S. aureus present in spiked milk. This ready-to-use device could be integrated with simple nucleic acid extraction for food pathogen detection in resource-limited settings.