Kevin M. Koo

DNA–bare gold affinity interactions: mechanism and applications in biosensing

The adsorption of DNA onto gold due to affinity interactions is highly desirable for developing low-cost, convenient and sensitive biosensors. To date, DNA–gold adsorption phenomenon has been demonstrated as one of the most promising physical mechanisms for achieving precise control over unmodified gold nanoparticles (AuNPs) aggregation, and DNA monolayer formation on gold surfaces. The adsorption phenomenon is exquisitely controlled by many factors including intermolecular forces, along with DNA composition and sequence. The understanding and manipulation of these factors have allowed broad biosensing applications and notably, sequence-dependent DNA–gold adsorption which may be highly relevant for DNA methylation detection in cancer. Herein, we review the underlying principles governing DNA–gold adsorption as well as recent biosensing strategies based on differential ssDNA/dsDNA–AuNPs adsorption and sequence-dependent DNA–gold adsorption. Finally, we have also contributed insights regarding the future trend of DNA–gold adsorption-based biosensors.

Microdevices for detecting locus-specific DNA methylation at CpG resolution

Simple, rapid, and inexpensive strategies for detecting DNA methylation could facilitate routine patient diagnostics. Herein, we describe a microdevice based electrochemical assay for the detection of locus-specific DNA methylation at single CpG dinucleotide resolution after bisulfite conversion of a target DNA sequence. This is achieved by using the ligase chain reaction (LCR) to recognize and amplify a C to T base change at a CpG site and measuring the change electrochemically (eLCR). Unlike other electrochemical detection methods for DNA methylation, methylation specific (MS)-eLCR can potentially interrogate any CpG of interest in the genome. MS-eLCR also distinguishes itself from other electrochemical LCR detection schemes by integrating a peroxidase-mimicking DNAzyme sequence into the LCR amplification probes design which in turn, serves as a redox moiety when bound with a hemin cofactor. Following hybridization to surface-bound capture probes, the DNAzyme-linked LCR products induce electrocatalytic responses that are proportional to the methylation levels of the gene locus in the presence of hydrogen peroxide. The performance of the assay was evaluated by simultaneously detecting C to T changes at a locus associated with cancer metastasis in breast cancer cell lines and serum-derived samples. MS-eLCR required as little as 0.04 pM of starting material and was sensitive to 10-15% methylation change with good reproducibility (RSD=7.9%, n=3). Most importantly, the accuracy of the method in quantifying locus-specific methylation was comparable to both fluorescence-based and Next Generation Sequencing approaches. We thus believe that the proposed assay could potentially be a low cost alternative to current technologies for DNA methylation detection of specific CpG sites.

Amplification-Free Detection of Gene Fusions in Prostate Cancer Urinary Samples Using mRNA–Gold Affinity Interactions

A crucial issue in present-day prostate cancer (PCa) detection is the lack of specific biomarkers for accurately distinguishing between benign and malignant cancer forms. This is causing a high degree of overdiagnosis and overtreatment of otherwise clinically insignificant cases. As around half of all malignant PCa cases display a detectable gene fusion mutation between the TMPRSS2 promoter sequence and the ERG coding sequence (TMPRSS2:ERG) in urine, noninvasive screening of TMPRSS2:ERG mRNA in patient urine samples could improve the specificity of current PCa diagnosis. However, current gene fusion detection methodologies are largely dependent on RNA enzymatic amplification, which requires extensive sample manipulation, costly labels for detection, and is prone to bias/artifacts. Herein we introduce the first successful amplification-free electrochemical assay for direct detection of TMPRSS2:ERG mRNA in PCa urinary samples by selectively isolating and adsorbing TMPRSS2:ERG mRNA onto bare gold electrodes without requiring any surface modification. We demonstrated excellent limit-of-detection (10 cells) and specificity using PCa cell line models, and showcased clinical utility by accurately detecting TMPRSS2:ERG in a collection of 17 urinary samples obtained from PCa patients. Furthermore, these results were validated with the current gold standard reverse transcription (RT)-PCR approach with 100% concordance.

Colorimetric TMPRSS2-ERG Gene Fusion Detection in Prostate Cancer Urinary Samples via Recombinase Polymerase Amplification.

TMPRSS2 (Exon 1)-ERG (Exon 4) is the most frequent gene fusion event in prostate cancer (PC), and is highly PC-specific unlike the current serum prostate specific antigen (PSA) biomarker. However, TMPRSS2-ERG levels are currently measured with quantitative reverse-transcription PCR (RT-qPCR) which is time-consuming and requires costly equipment, thus limiting its use in clinical diagnostics. Herein, we report a novel rapid, cost-efficient and minimal-equipment assay named “FusBLU” for detecting TMPRSS2-ERG gene fusions from urine. TMPRSS2-ERG mRNA was amplified by isothermal reverse transcription-recombinase polymerase amplification (RT-RPA), magnetically-isolated, and detected through horseradish peroxidase (HRP)-catalyzed colorimetric reaction. FusBLU was specific for TMPRSS2-ERG mRNA with a low visual detection limit of 105 copies. We also demonstrated assay readout versatility on 3 potentially useful platforms. The colorimetric readout was detectable by naked eye for a quick yes/no evaluation of gene fusion presence. On the other hand, a more quantitative TMPRSS2-ERG detection was achievable by absorbance/electrochemical measurements. FusBLU was successfully applied to 12 urinary samples and results were validated by gold-standard RT-qPCR. We also showed that sediment RNA was likely the main source of TMPRSS2-ERG mRNA in urinary samples. We believe that our assay is a potential clinical screening tool for PC and could also have wide applications for other disease-related fusion genes.

A simple, rapid, low-cost technique for naked-eye detection of urine-isolated TMPRSS2:ERG gene fusion RNA

The TMPRSS2:ERG gene fusion is one of a series of highly promising prostate cancer (PCa) biomarker alternatives to the controversial serum PSA. Current methods for detecting TMPRSS2:ERG are limited in terms of long processing time, high cost and the need for specialized equipment. Thus, there is an unmet need for less complex, faster, and cheaper methods to enable gene fusion detection in the clinic. We describe herein a simple, rapid and inexpensive assay which combines robust isothermal amplification technique with a novel visualization method for evaluating urinary TMPRSS2:ERG status at less than USD 5 and with minimal equipment. The assay is sensitive, and rapidly detects as low as 105 copies of TMPRSS2:ERG transcripts while maintaining high levels of specificity.

High-speed biosensing strategy for non-invasive profiling of multiple cancer fusion genes in urine

Aberrant chromosal rearrangements, such as the multiple variants of TMPRSS2:ERG fusion gene mutations in prostate cancer (PCa), are promising diagnostic and prognostic biomarkers due to their specific expression in cancerous tissue only. Additionally, TMPRSS2:ERG variants are detectable in urine to provide non-invasive PCa diagnostic sampling as an attractive surrogate for needle biopsies. Therefore, rapid and simplistic assays for identifying multiple urinary TMPRSS2:ERG variants are potentially useful to aid in early cancer detection, immediate patient risk stratification, and prompt personalized treatment. However, current strategies for simultaneous detection of multiple gene fusions are limited by tedious and prolonged experimental protocols, thus limiting their use as rapid clinical screening tools. Herein, we report a simple and rapid gene fusion strategy which expliots the specificity of DNA ligase and the speed of isothermal amplification to simultaneously detect multiple fusion gene RNAs within a short sample-to-answer timeframe of 60min. The method has a low detection limit of 2 amol (1000 copies), and was successfully applied for non-invasive fusion gene profiling in patient urine samples with subsequent validation by a PCR-based gold standard approach.

DNA-directed assembly of copper nanoblocks with inbuilt fluorescent and electrochemical properties: Application in simultaneous amplification-free analysis of multiple RNA species

The intrinsic affinity of DNA molecules toward metallic ions can drive the specific formation of copper nanostructures within the nucleic acid helix structure in a sequence-dependent manner. The resultant nanostructures have interesting fluorescent and electrochemical properties, which are attractive for novel biosensing applications. However, the potential of using DNA-templated nanostructures for precision disease diagnosis remains unexplored. Particularly, DNAtemplated nanostructures show high potential for the universal amplification-free detection of different RNA biomarker species. Because of their low cellular levels and differing species-dependent length and sequence features, simultaneous detection of different messenger RNAs, microRNAs, and long non-coding RNAs species with a single technique is challenging. Here, we report a contemporary technique for facile in situ assembly of DNA-templated copper nanoblocks (CuNBs) on various RNA species targets after hybridization-based magnetic isolation. Our approach circumvents the typical limitations associated with amplification and labeling procedures of current RNA assays. The synthesized CuNBs enabled amplification-free fM-level RNA detection with flexible fluorescence or electrochemical readouts. Furthermore, our nanosensing technique displays potential for clinical application, as demonstrated by non-invasive analysis of three diagnostic RNA biomarkers from a cohort of 10 prostate cancer patient urinary samples with 100%-concordance (quantitative reverse transcriptionpolymerase chain reaction (PCR) validation). The good analytical performance and versatility of our method may be useful in both diagnostics and research fields.

Amplification‐Free Multi‐RNA‐Type Profiling for Cancer Risk Stratification via Alternating Current Electrohydrodynamic Nanomixing

Simultaneous analysis of messenger RNA (mRNA), microRNA (miRNA), and long noncoding RNA (lncRNA)-multi-RNA-type profiling-is increasingly crucial in cancer diagnostics. Yet, rapid multi-RNA-type profiling is challenging due to enzymatic amplification reliance and RNA-type-dependent characteristics. Here, a nanodevice is reported to uniquely use alterable alternating current electrohydrodynamic (ac-EHD) forces to enhance probe-target hybridization prior to direct native RNA target detection, without target amplification or surface functionalization. To exemplify clinical applicability, noninvasive screening of next-generation prostate cancer (PCa) RNA biomarkers (of different types) in patient urine samples is performed. A strong correlation between multi-RNA-type expression and aggressive PCa is found, and the nanodevice performance is statistically evaluated. It is believed that this miniaturized system exhibits great potential for cancer risk stratification via multi-RNA-type profiling.

Design and Clinical Verification of Surface-Enhanced Raman Spectroscopy Diagnostic Technology for Individual Cancer Risk Prediction

The use of emerging nanotechnologies, such as plasmonic nanoparticles in diagnostic applications, potentially offers opportunities to revolutionize disease management and patient healthcare. Despite worldwide research efforts in this area, there is still a dearth of nanodiagnostics which have been successfully translated for real-world patient usage due to the predominant sole focus on assay analytical performance and lack of detailed investigations into clinical performance in human samples. In a bid to address this pressing need, we herein describe a comprehensive clinical verification of a prospective label-free surface-enhanced Raman scattering (SERS) nanodiagnostic assay for prostate cancer (PCa) risk stratification. This contribution depicts a roadmap of (1) designing a SERS assay for robust and accurate detection of clinically validated PCa RNA targets; (2) employing a relevant and proven PCa clinical biomarker model to test our nanodiagnostic assay; and (3) investigating the clinical performance on independent training ( n = 80) and validation ( n = 40) cohorts of PCa human patient samples. By relating the detection outcomes to gold-standard patient biopsy findings, we established a PCa risk scoring system which exhibited a clinical sensitivity and specificity of 0.87 and 0.90, respectively [area-under-curve of 0.84 (95% confidence interval: 0.81-0.87) for differentiating high- and low-risk PCa] in the validation cohort. We envision that our SERS nanodiagnostic design and clinical verification approach may aid in the individualized prediction of PCa presence and risk stratification and may overall serve as an archetypical strategy to encourage comprehensive clinical evaluation of nanodiagnostic innovations.

Interfacial Biosensing: Direct Biosensing of Biomolecules at the Bare Metal Interface

Interfacial biosensing is an emerging research field which harnesses the differential adsorption interactions of biological species (e.g., DNA, RNA, proteins) with bare metal surfaces for direct biomolecule detection and analysis. During disease progression, biomolecules commonly undergo considerable changes in their molecular structure and three-dimensional conformation, as compared to their normal states. This can significantly alter the biomolecules’ physicochemical properties to affect adsorption interactions with metallic surfaces. Although differential adsorption of molecules on nonmetallic surfaces has long been used for chromatographic separation, the biosensing application of this phenomenon on metallic surfaces has never been considered till recent times. Interfacial biosensing innovatively exploits the adsorption behavior of biomolecules in a novel fashion (via direct biomolecule-metal surface interactions) to enable a new age of clinical translation-directed technologies for genomic, transcriptomic, and proteomic analysis. Specifically, it is envisioned that interfacial biosensing can overcome major technological drawbacks of current biosensing approaches by (i) obviating the need for surface biofunctionalization steps and (ii) significantly reducing the analysis time and assay cost. This review will focus on recent advances and prospects of interfacial biosensing and also highlight the most significant applications developed by current research.