Colorimetric molecular diagnosis of the HIV gag gene using DNAzyme and a complementary DNA-extended primer.

We have developed a novel strategy for the colorimetric detection of PCR products by utilizing a target-specific primer modified at the 5'-end with an anti-DNAzyme sequence. A single-stranded DNAzyme sequence folds into a G-quadruplex structure with hemin and shows strong peroxidase activity. When the complementary strand binds to the DNAzyme sequence, it blocks the formation of the G-quadraduplex structure and loses its peroxidase activity. In the presence of the target gene, PCR amplification proceeds, and anti-DNAzyme sequence modified primers present in the reaction mixture form a double strand through primer extension. Therefore, it does not block the DNAzyme sequence. Further, a colorimetric signal is generated by the addition of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and H2O2 at the end of the reaction. We have successfully detected a single copy of the HIV type 1 gag gene in buffer and 10 copies in human serum. The strategy developed could be used to detect DNA and RNA in complex biological samples by simple primer designing that includes DNAzyme and a DNA extended primer.

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.

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.

Lab-on-paper for all-in-one molecular diagnostics (LAMDA) of zika, dengue, and chikungunya virus from human serum.

Several tropical fever viruses transmitted by mosquitoes including zika, dengue, and chikungunya, are becoming a serious problem in global public health. Simple diagnostic tools in early stages are strongly required to monitor and prevent these diseases. Paper diagnostic platforms can provide a solution for these needs, with integration of fluidic control techniques and isothermal amplification methods. Here, we demonstrate a Lab-on-paper for all-in-one molecular diagnostics of zika, dengue, and chikungunya virus from human serum. The entire process of nucleic acid testing that involves sampling, extraction, amplification, and detection is simply operated on a single paper chip. Based on the engineered structure of paper materials and dried chemicals on the all-in-one chip, serum samples containing the target virus RNA were simply added by automatic flow from distilled water injection. Target RNA molecules were concentrated on the binding pad with chitosan and then transported to reaction pads following a pH increase for specific reverse transcription loop-mediated isothermal amplification with fluorescence signal generation. Three targets, zika virus, dengue virus, and chikungunya virus, in human serum were simultaneously detected on the all-in-one paper chip within 60 min at 65 °C. The all-in-one paper chip can be used as a real-time quantitative assay for 5-5000 copies of zika virus RNA. This all-in-one device was successfully used with 5 clinical specimens of zika and dengue virus from real patients. We believe that the proposed all-in-one paper chip can provide a portable, low-cost, user-friendly, sensitive, and specific NAT platform with great potential in point-of-care diagnostics.

An innovative paper-based device for DNA extraction from processed meat products.

Detection of food adulteration is a challenge. However, the identification of adulterated meat in processed products is important for health and personal preference. Mitochondrial genomic DNA (mtDNA) is a good candidate for reliable identification of meat ingredients; however, the extraction of mtDNA from processed products is a bottleneck for development of detection strategies. Therefore, we constructed a rapid (~5 min) mtDNA extraction device. mtDNAs from different meat samples, such as pork (Sus scrofa), chicken (Gallus gallus), and beef (Bos taurus), were successfully detected in up to 0.1% adulterated animal species. We believe that the proposed strategy could be applied to detect animal species from processed meat products to reduce fraudulent practices.

Paper-based nucleic acid testing system for simple and early diagnosis of mosquito-borne RNA viruses from human serum.

The recent outbreaks of mosquito-borne diseases (e.g., zika, dengue, and chikungunya) increased public health burden in developing countries. To control the spread of these infectious diseases, a simple, economic, reliable, sensitive, and selective diagnostic platform is required. Considering demand for affordable and accessible methods, we have demonstrated a two-step strategy for extraction and detection of viral RNAs of infectious diseases within 1 h. Ready-to-use devices for viral RNA extraction and detection were successfully fabricated using paper as a substrate. Viral RNA (e.g., zika, dengue, and chikungunya) was captured and eluted using a handheld RNA extraction paper-strip device, and another paper-chip device was used for reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with a detection limit of a single copy and 10 copies of viral RNA in phosphate buffer solution (PBS) and serum, respectively. With these proposed devices, we have detected viral RNAs of zika and dengue in clinical human serum samples. The proposed paper-based extraction and detection platforms could be employed for detection of infectious viral diseases from complex clinical samples in resource-limited settings.

Polymerization-sensitive switch-on monomer for terminal transferase activity assay.

We herein describe a simple but efficient method for the determination of terminal transferase (TdT) activity, which relies on our finding that Fe(III)-quenched boron-dipyrromethene (BODIPY)-ATP is utilized as a switch-on monomer for polymerization and enables the facile synthesis of fluorescence oligonucleotides without additional, post-processing steps. As TdT carries out the synthesis of DNA by adding the monomers into growing chains, Fe(III) is displaced from BODIPY with the release of pyrophosphate group, which consequently leads to the generation of highly fluorescent long oligonucleotides. With this strategy, we selectively detected the TdT activity with high sensitivity. In addition, its practical applicability was successfully demonstrated by determining TdT activities in human serum.

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.

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.

Novel amine-functionalized iron trimesates with enhanced peroxidase-like activity and their applications for the fluorescent assay of choline and acetylcholine.

We herein describe novel amine-grafted metal-organic frameworks (MOFs) as a promising alternative to natural peroxidase enzyme and their applications for a fluorescent assay of choline (Cho) and acetylcholine (ACh). Among diverse amine-functionalized MOFs, N,N,N',N'-tetramethyl-1,4-butanediamine (TMBDA)-functionalized MIL-100(Fe) (TMBDA-MIL-100(Fe)) exhibited the highest peroxidase activity by developing intense fluorescence from Amplex UltraRed (AUR) in the presence of H2O2, which was presumably due to the synergetic effect of the enhanced negative potential and precisely controlled molecular size of the grafted diamine. Based on the excellent peroxidase-like activity of TMBDA-MIL-100(Fe), choline and ACh were reliably determined down to 0.027 and 0.036µM, respectively. Furthermore, practical applicability of this strategy was successfully demonstrated by detecting choline and ACh in spiked samples of milk and serum, respectively. This work highlights the advantages of amine-grafted MOFs for the preparation of biomimetic catalysts, extending their scope to biosensor applications.

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.

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.