Hierarchical integration of DNA nanostructures and NanoGold onto a microchip facilitates covalent chemistry-mediated purification of circulating tumor cells in head and neck squamous cell carcinoma.

It is well-established that the combined use of nanostructured substrates and immunoaffinity agents can enhance the cell-capture performance of the substrates, thus offering a practical solution to effectively capture circulating tumor cells (CTCs) in peripheral blood. Developing along this strategy, this study first demonstrated a top-down approach for the fabrication of tetrahedral DNA nanostructure (TDN)-NanoGold substrates through the hierarchical integration of three functional constituents at various length-scales: a macroscale glass slide, sub-microscale self-organized NanoGold, and nanoscale self-assembled TDN. The TDN-NanoGold substrates were then assembled with microfluidic chaotic mixers to give TDN-NanoGold Click Chips. In conjunction with the use of copper (Cu)-catalyzed azide-alkyne cycloaddition (CuAAC)-mediated CTC capture and restriction enzyme-triggered CTC release, TDN-NanoGold Click Chips allow for effective enumeration and purification of CTCs with intact cell morphologies and preserved molecular integrity. To evaluate the clinical utility of TDN-NanoGold Click Chips, we used these devices to isolate and purify CTCs from patients with human papillomavirus (HPV)-positive (+) head and neck squamous cell carcinoma (HNSCC). The purified HPV(+) HNSCC CTCs were then subjected to RT-ddPCR testing, allowing for detection of E6/E7 oncogenes, the characteristic molecular signatures of HPV(+) HNSCC. We found that the resulting HPV(+) HNSCC CTC counts and E6/E7 transcript copy numbers are correlated with the treatment responses in the patients, suggesting the potential clinical utility of TDN-NanoGold Click Chips for non-invasive diagnostic applications of HPV(+) HNSCC.

Target-induced transcription of a light-up RNA aptamer to construct a novel method for alkaline phosphatase assay.

We herein developed a novel method for alkaline phosphatase (ALP) assay based on the target-induced transcription of a light-up RNA aptamer, consequently producing a highly enhanced fluorescence signal upon specifically binding to the corresponding dye. Using this strategy, we successfully determined the ALP activity down to 0.018 U L-1 (dynamic linear range of 0.04-4 U L-1) with excellent selectivity.

Ultrasensitive version of nucleic acid sequence-based amplification (NASBA) utilizing a nicking and extension chain reaction system.

Nucleic acid sequence-based amplification (NASBA) is a transcription-based isothermal amplification technique especially designed for the detection of RNA targets. The NASBA basically relies on the linear production of T7 RNA promoter-containing double-stranded DNA (T7DNA), and thus the final amplification efficiency is not sufficiently high enough to achieve ultrasensitive detection. We herein ingeniously integrate a nicking and extension chain reaction system into the NASBA to establish an ultrasensitive version of NASBA, termed Nicking and Extension chain reaction System-Based Amplification (NESBA). By employing a NESBA primer set designed to contain an additional nicking site at the 5' end of a NASBA primer set, the T7DNA is exponentially amplified through continuously repeated nicking and extension chain reaction by the combined activities of nicking endonuclease (NE) and reverse transcriptase (RT). As a consequence, a much larger number of RNA amplicons would be produced through the transcription of the amplified T7DNA, greatly enhancing the final fluorescence signal from the molecular beacon (MB) probe binding to the RNA amplicon. Based on this unique design principle, we successfully identified the target respiratory syncytial virus A (RSV A) genomic RNA (gRNA) down to 1 aM under isothermal conditions, which is 100-fold more sensitive than regular NASBA.

A novel method to detect mutation in DNA by utilizing exponential amplification reaction triggered by the CRISPR-Cas9 system.

We, herein, describe a novel method to detect mutation in DNA by utilizing exponential amplification reaction (EXPAR) triggered by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, called CRISPR-EXPAR. The CRISPR system consisting of two Cas9/sgRNA complexes was designed to cut out a specific mutation region within the target DNA, which would consequently promote EXPAR by continuously repeated extension and nicking reactions. As a consequence, a large number of final EXPAR products, which can be monitored through duplex-specific fluorescent staining, are produced. Based on this design principle, we successfully identified a model target mutation within the human epidermal growth factor receptor 2 (HER2) gene down to 437 aM with excellent specificity. The practical capability of this method was verified by reliably identifying the target mutation directly from the genomic DNA (gDNA) extracted from the lung cancer cell line, NCI-H1781 (H1781), and its universal applicability was further confirmed by identifying another EFGF L858R mutation. This technique could serve as a new isothermal platform to identify various mutations by rationally redesigning single guide RNA (sgRNA) according to the target mutation site.

Self-priming phosphorothioated hairpin-mediated isothermal amplification.

We herein describe a novel technology, termed self-priming phosphorothioated hairpin-mediated isothermal amplification (SP-HAMP), enabling target nucleic acid detection. Isothermal amplification strategies are a simple process that efficiently raises the amount of nucleic acid at a constant temperature, but still has lots of problems such as the requirement of multiple exogenous primers and enzymes, which trigger non-specific background signal and increase the complexity of procedures. The key component for overcoming the above-mentioned limitations is the designed hairpin probe (HP) consisting of self-priming region along the 3' stem and the 3' overhang and phosphorothioate modifications at the 5' overhang and the specific loop part. The HP was designed to open through binding to target nucleic acid. Upon opening of HP, its self-priming (SP) region is rearranged to form a smaller hairpin whose 3' end could serve as a primer. The following extension produces the extended HP and displaces the bound target nucleic acid, which is then recycled to open another HP. Due to the reduced stability caused by the specific two phosphorothioate (PS) modifications, the 3' end of EP1 is readily rearranged to form the foldback hairpin structure, which would promote the foldback extension to produce once more extended HP. Since the two PS modifications are always located at the same positions along the 5' stem within the further extended HPs, the foldback reaction followed by the extension would be continuously repeated, consequently producing a large number of the long hairpin concatamers. Based on this unique design principle, we successfully detected even a single copy of target DNA with outstanding discrimination capability under an isothermal condition by employing only a single HP without the requirement for the complicated multiple primers. In conclusion, the sophisticated design principle employed in this work would provide great insight for the development of self-operative isothermal amplifying system enabling short target nucleic acid detection such as microRNAs or any target which is less than 200 mer.

Colorimetric detection of individual biothiols by tailor made reactions with silver nanoprisms.

We herein described a rapid, sensitive, and selective colorimetric sensing platform for biothiols in human serum, which relies on the dual functions of biothiols as anti-etching and aggregating agent for silver nanoprisms (AgNPRs). In principle, the target biothiols that bind to the surface of AgNPRs through Ag-S covalent interactions protect the AgNPRs from being etched by chloride ion (Cl-) in human serum, thus exhibiting the blue/purple color that is indicative of AgNPRs. On the other hand, the color of AgNPRs turned to yellow in the absence of biothiols or the presence of non-sulfur-containing amino acids, indicating the formation of small silver nanoparticles (AgNPs). Importantly, we found that individual biothiols (Hcy, Cys, and GSH) exert not only the anti-etching effect, but also the aggregating effect on AgNPRs, which can be modulated by simply tuning the pH conditions, and this consequently allows for the discriminative detection of each biothiol. Based on this simple and cost-effective strategy, we successfully determined the Hcy, Cys, and GSH in human serum with high sensitivity and selectivity within 10 min, demonstrating the diagnostic capability and potential in practical applications.

Ultrasensitive isothermal method to detect microRNA based on target-induced chain amplification reaction.

We herein describe an ultrasensitive isothermal method to detect microRNA (miRNA) by utilizing target-induced chain amplification reaction (CAR). The hairpin probe (HP) employed in this strategy is designed to be opened upon binding to target miRNA. The exponential amplification reaction (EXPAR) template (ET) then binds to the exposed stem of HP and DNA polymerase (DP) promotes the extension reactions for both HP and ET, consequently producing intermediate double-stranded DNA product (IP) and concomitantly recycling target miRNA to open another intact HP. The IPs would produce a large number of target-mimicking probes (TMPs) and trigger probes (TPs) through the continuously repeated nicking and extension reactions at the two separated nicking sites within the IP. TMP triggers another CAR cycle by binding to intact HP as target miRNA did while TP promotes conventional EXPAR by independently binding to free ET. As a consequence of these interconnected reaction systems, a large number of final double-stranded DNA products (FPs) are produced, which can be monitored by measuring the fluorescent signal produced from duplex-specific fluorescent dye. Based on this unique design principle, the target miRNA was successfully determined down to even a single copy with high selectivity against non-specific miRNAs. The practical applicability of this method was also verified by reliably detecting target miRNA included in the total RNA extracted from the human cancer cell.

Portable glucose meter-utilized label-free and washing-free telomerase assay.

We herein describe a portable glucose meter (PGM)-utilized label-free and washing-free method for the facile determination of telomerase activity that relies on the kinase-catalyzed cascade enzymatic reaction (KCER) that transduces the telomerase activity to the glucose level. In the sensor, the telomerase that elongates telomere sequences ((TTAGGG)n) from the 3'-terminus of telomerase substrate primer (TSP) consumes deoxynucleoside triphosphate (dNTP), which serves as a phosphate source for KCER promoted by hexokinase and pyruvate kinase. Thus, the presence of telomerase protects KCER from working effectively, resulting in the maintenance of an initial, high glucose level that is readily determined using hand-held PGM. With this strategy, the telomerase activities in various types of cell lines were successfully determined with high sensitivity. Furthermore, the ability of this method to screen candidate inhibitors for telomerase activity was also verified.

Simple and label-free strategy for terminal transferase assay using a personal glucose meter.

We herein developed a simple personal glucose meter (PGM)-based method for terminal transferase (TdT) activity assay by utilizing the glucose oxidase (GOx)-mimicking activity of cerium oxide nanoparticles (CeO2 NPs). Using this strategy, the TdT activity was reliably determined down to 0.7 U mL-1 with high selectivity against other non-specific enzymes.

A hairpin probe-mediated isothermal amplification method to detect target nucleic acid.

We herein describe Hairpin probe-mediated Isothermal Amplification (HIAmp), a novel isothermal method to detect a target nucleic acid. This method employs a hairpin probe (HP) designed to be opened through binding to the target nucleic acid. Upon opening of the HP, the primer binds to the free stem of the opened HP followed by its extension by DNA polymerase, consequently displacing and recycling the target nucleic acid to open another HP and producing an intermediate product (IP) containing a nicking site. The IP then continuously produces a trigger probe (TP), which subsequently initiates the isothermal amplification cycles in two separate ways by binding to either the intact HP or the overhang region of the IP. Through the following well-designed interconnected pathways, a large amount of final double-stranded DNA products (FPs) is produced and a high fluorescent signal is generated from the duplex-specific fluorescent dye, SYBR Green I. By employing this isothermal strategy, target DNA was very sensitively detected down to 64 zmol with the capability to discriminate the target DNA against non-specific DNAs. This work would provide remarkable insight into the design of a new DNA network enabling isothermal amplification.

Glucose oxidase-like activity of cerium oxide nanoparticles: use for personal glucose meter-based label-free target DNA detection.

Recently, personal glucose meter (PGM) has been utilized for the detection of non-glucose targets for point-of-care (POC) testing. Aimed at this goal, we herein developed a new PGM-based label-free read-out method for polymerase chain reaction (PCR) based on our novel finding that cerium oxide nanoparticles (CeO2 NPs) exhibit glucose oxidase-like activity comparable to the natural glucose oxidase enzyme. Methods: In principle, DNA amplicons produced by PCR in the presence of target DNA electrostatically bind to CeO2 NPs, leading to their aggregation and reducing the efficiency for CeO2 NP-catalyzed glucose oxidation reaction. Thus, glucose is hardly oxidized to gluconic acid, resulting in the maintenance of initial high glucose level. On the contrary, in the absence of target DNA or presence of non-target DNA, DNA amplicons are not produced and glucose is effectively oxidized by the glucose oxidase-like activity of CeO2 NPs, leading to the significant reduction of glucose level. Finally, the resulting glucose level is simply measured by using PGM. Results: With this strategy, DNA amplicons were quantitatively examined within 5 min, realizing ultrafast analysis of PCR results without any cumbersome and labor-intensive procedures. In addition, the target genomic DNA derived from Escherichia coli (E. coli) was sensitively determined down to 10 copies with high selectivity. Conclusion: Importantly, the use of PGM as a detection component enables its direct application in POC settings. Based on the meritorious features of PGM such as rapidity, simplicity, and cost-effectiveness, we expect that the devised system could serve as a core platform for the on-site read-out of PCR amplification.

Three-way junction-induced isothermal amplification for nucleic acid detection.

We, herein, describe a three-way junction (3WJ)-induced isothermal amplification (ThIsAmp) reaction for target nucleic acid detection. In this strategy, target nucleic acid induces the formation of 3WJ structure by associating a specially designed ThIsAmp template and ThIsAmp primer. Upon the formation of 3WJ structure, ThIsAmp primer is subjected to continuously repeated extension and nicking reaction by the combined activities of DNA polymerase and nicking endonuclease, consequently producing a large number of trigger strands. The trigger strands then initiate two separate but interconnected pathways by binding to either 3' overhang of ThIsAmp template within the 3WJ structure or free ThIsAmp template. As a consequence, a large number of final double-stranded DNA products are produced under an isothermal condition, which can be monitored in real-time by detecting the fluorescence intensity resulting from SYBR Green I staining. Based on this principle, we successfully detected target DNA down to 78.1 aM with excellent specificity. The sophisticated design principle employed in this work would provide great insight for the development of self-operative isothermal amplifying system enabling target nucleic acid detection.

Label-free and washing-free alkaline phosphatase assay using a personal glucose meter.

We herein describe a personal glucose meter (PGM)-based method for a label-free and washing-free determination of alkaline phosphatase (ALP) activity, which relies on the cascade enzymatic reactions promoted by hexokinase and pyruvate kinase to couple ALP activity with the amount of glucose. In principle, the presence of target ALP scavenges on adenosine 5'-triphosphate (ATP), a phosphate source for hexokinase-catalyzed reactions, and thus suppresses the ensuing cascade enzymatic reactions. As a result, the initial high amount of glucose is maintained and the amount of glucose, which is proportional to ALP activity, is simply measured by a hand-held PGM. Based on this novel strategy, we successfully determined the ALP activity down to 8.9 U/L with the high selectivity. In addition, the diagnostic capability of this method was demonstrated by reliably assaying the ALP activity in non-diluted human blood without any pretreatment steps.

Fluorescent S1 nuclease assay utilizing exponential strand displacement amplification.

We herein devise a simple and label-free strategy to determine S1 nuclease activity by exploiting the target-induced inhibition of exponential strand displacement amplification (eSDA). In principle, a DNA probe that is designed to produce a large amount of duplexes through a process of eSDA, is degraded by the catalytic activity of S1 nuclease. This reaction blocks the initiation of eSDA, leading to the quite-reduced fluorescence of a double-stranded DNA specific fluorescent dye, SYBR Green I compared to the one in the absence of S1 nuclease. With this simple but novel approach, the S1 nuclease activity was selectively assayed with the high sensitivity. In addition, this system was successfully demonstrated to possess the capability to screen potential inhibitors against S1 nuclease.

A Personal Glucose Meter for Label-Free and Washing-Free Biomolecular Detection.

We developed a label-free and washing-free method for biomolecular detection using a personal glucose meter (PGM). ATP was selected as a model target, and cascade enzymatic reactions promoted by hexokinase and pyruvate kinase were adopted to link the amount of ATP to glucose that is detectable by a hand-held PGM. In principle, the presence of target ATP enables hexokinase to catalyze the conversion of glucose to glucose 6-phosphate by providing a phosphate group to glucose, and thus the amount of glucose is decreased in proportion to the amount of ATP. In addition, adenosine 5'-diphosphate (ADP), which is generated after hexokinase-catalyzed enzymatic reaction, is recovered to ATP by a pyruvate kinase enzyme. The regenerated ATP is again supplemented to catalyze multiple rounds of cascade enzymatic reactions, leading to signal amplification. As a result, the change of glucose amount that is inversely proportional to ATP amount is simply measured by a hand-held PGM. By employing this strategy, we successfully determined ATP down to 49 nM with high selectivity even in real samples such as tap water, human serum, and bovine urine. Importantly, the developed system does not require expensive modification and washing steps but is conveniently operated with a commercially available PGM, which would pave the way for the development of a simple and cost-effective sensing platform.

A simple and sensitive detection of small molecule-protein interactions based on terminal protection-mediated exponential strand displacement amplification.

We herein describe a simple and sensitive strategy to detect a small molecule-protein interaction based on terminal protection-mediated exponential strand displacement amplification (eSDA). In principle, the small molecule linked to a DNA probe protects the DNA probe against the exonuclease I-catalyzed degradation after its binding to the corresponding target protein. The protected DNA probe then serves as a template to promote eSDA. Consequently, a large number of duplexes are produced, which leads to a high fluorescence from a double-stranded DNA specific fluorescent dye, SYBR Green I. As a model system to prove this sensing strategy, the interaction between biotin and streptavidin (SA), which is known to be the strongest among the non-covalent biological interactions, was selected and its analytical performance was thoroughly investigated. As a result, SA was sensitively detected with the limit of detection of 16 pM. In addition, the practical applicability of this method was successfully demonstrated by reliably determining the SA in human serum.

Universally applicable, quantitative PCR method utilizing fluorescent nucleobase analogs.

We herein describe a novel quantitative PCR (qPCR) method, which operates in both signal-off and on manners, by utilizing a unique property of fluorescent nucleobase analogs. The first, signal-off method is developed by designing the primers to contain pyrrolo-dC (PdC), one of the most common fluorescent nucleobase analogs. The specially designed single-stranded primer is extended to form double-stranded DNA during PCR and the fluorescence signal from the PdCs incorporated in the primer is accordingly reduced due to its conformation-dependent fluorescence properties. In addition, the second, signal-on method is devised by designing the primers to contain 5'-overhang sequences complementary to the PdC-incorporated DNA probes. At the initial phase, the PdC-incorporated DNA probes are hybridized to the 5'-overhang sequences of the primer, exhibiting the significantly quenched fluorescence signal, but are detached by either hydrolysis or strand displacement reaction during PCR, leading to the highly enhanced fluorescence signal. This method is more advanced than the first one since it produces signal-on fluorescence response and permits the use of a single PdC-incorporated DNA probe for the detection of multiple target nucleic acids, remarkably decreasing the assay cost. With these novel qPCR methods, we successfully quantified target nucleic acids derived from sexually transmitted disease (STD) pathogens with high accuracy. Importantly, the proposed strategies overcome the major drawbacks in the current SYBR Green and TaqMan probe-based qPCR methods such as low specificity and high assay cost.

A new s-adenosylhomocysteine hydrolase-linked method for adenosine detection based on DNA-templated fluorescent Cu/Ag nanoclusters.

We herein describe a novel fluorescent method for the rapid and selective detection of adenosine by utilizing DNA-templated Cu/Ag nanoclusters (NCs) and employing s-adenosylhomocysteine hydrolase (SAHH). SAHH is allowed to promote hydrolysis reaction of s-adenosylhomocysteine (SAH) and consequently produces homocysteine, which would quench the fluorescence signal from DNA-templated Cu/Ag nanoclusters employed as a signaling probe in this study. On the other hand, adenosine significantly inhibits the hydrolysis reaction and prevent the formation of homocysteine. Consequently, highly enhanced fluorescence signal from DNA-Cu/Ag NCs is retained, which could be used to identify the presence of adenosine. By employing this design principle, adenosine was sensitively detected down to 19nM with high specificity over other adenosine analogs such as AMP, ADP, ATP, cAMP, guanosine, cytidine, and urine. Finally, the diagnostic capability of this method was successfully verified by reliably detecting adenosine present in a real human serum sample.

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.