Paper-Based Radial Flow Assay Integrated to Portable Isothermal Amplification Chip Platform for Colorimetric Detection of Target DNA

김태용,Kim Sanha,Jung Jae Hwan,우민아 한국바이오칩학회 2023 BioChip Journal Vol.17 No.2

A novel integrated detection system that introduces a paper-chip-based molecular detection strategy into a polydimethylsiloxane (PDMS) microchip and temperature control system was developed for on-site colorimetric detection of DNA. For the paper chip-based detection strategy, a padlock probe DNA (PLP)-mediated rolling circle amplification (RCA) reaction for signal amplification and a radial flow assay according to the Au-probe labeling strategy for visualization were optimized and applied for DNA detection. In the PDMS chip, the reactions for ligation of target-dependent PLP, RCA, and labeling were performed one-step under isothermal temperature in a single chamber, and one drop of the final reaction solution was loaded onto the paper chip to form a radial colorimetric signal. To create an optimal analysis environment, not only the optimization of molecular reactions for DNA detection but also the chamber shape of the PDMS chip and temperature control system were successfully verified. Our results indicate that a detection limit of 14.7 nM of DNA was achieved, and non-specified DNAs with a single-base mismatch at the target DNA were selectively discriminated. This integrated detection system can be applied not only for single nucleotide polymorphism identification, but also for pathogen gene detection. The adoption of inexpensive paper and PDMS chips allows the fabrication of cost-effective detection systems. Moreover, it is very suitable for operation in various resource-limited locations by adopting a highly portable and user-friendly detection method that minimizes the use of large and expensive equipment.

One-step DNA purification and amplification on an integrated plastic microdevice for on-site identification of foodborne pathogens

Trinh, Kieu The Loan,Zhang, Yu,Lee, Nae Yoon Elsevier 2018 Analytica chimica acta Vol.1040 No.-

<P><B>Abstract</B></P> <P>In this study, we demonstrate a simple and facile method to enable DNA purification and amplification in a continuous step inside a thermoplastic microdevice. The innate property of thermoplastics was adopted to simplify DNA purification because negatively charged DNA can electrostatically interact with the induced positively charged surfaces of thermoplastics as a sample flows inside a microchannel; thus, the DNA purification step can be eliminated. We report the use of natural plastics for the selective adsorption of DNA and conduct a subsequent flow-through polymerase chain reaction (PCR) using a thermoplastic microdevice. Four thermoplastics—poly(methyl methacrylate) (PMMA), polycarbonates (PC), polystyrene (PS), and polypropylene (PP)—were selected as analytical models, and DNA adsorption phenomena on their pristine surfaces were examined. DNA was successfully immobilized onto the pristine thermoplastic surfaces, and the results were confirmed by fluorescent measurement and contact angle measurement and by performing PCR using a thermocycler. The electrostatically attached DNA was subsequently released from the pristine thermoplastic surface using a PCR reagent, which contained a low ionic strength salt as a component that could be used for DNA elution. The eluted DNA was then seamlessly amplified in the subsequent microchannel designed to perform flow-through PCR. The PCR products were collected by a microchamber connected at the end of the microchannel for on-site fluorescence detection. The PMMA microdevice was used to successfully purify and amplify a 210 bp target amplicon from a foodborne pathogen, <I>Escherichia coli</I> O157:H7, within 35 min. This study is expected to pave the way for developing an integrated monolithic thermoplastic microdevice suitable for rapidly identifying foodborne pathogens with reduced operation, decreased manufacturing cost, and enhanced device disposability.</P> <P><B>Highlights</B></P> <P> <UL> <LI> An integrated monolithic PMMA device was fabricated for pathogens identification. </LI> <LI> Plastic surface itself was utilized for DNA attachment for sample purification. </LI> <LI> The eluted DNA by PCR reagents was successfully amplified inside microchannel. </LI> <LI> DNA purification, amplification, and detection were realized in a seamless manner. </LI> <LI> The overall process for foodborne pathogen detection was realized within 35 min. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>We fabricate a fully integrated monolithic PMMA microdevice and demonstrate DNA purification, amplification, and on-site detection of major foodborne pathogens in one continuous flow.</P> <P>[DISPLAY OMISSION]</P>

Liquid coplanar-gate organic/graphene hybrid electronics for label-free detection of single and double-stranded DNA molecules

Kim, Jin Woo,Jang, Yoon-ha,Ku, Gwang Mo,Kim, Seunghyun,Lee, Eunho,Cho, Kilwon,Lim, Kwang-il,Lee, Wi Hyoung Elsevier 2018 Organic Electronics Vol.62 No.-

<P><B>Abstract</B></P> <P>The label-free detection of DNA with simple device structure and materials helps rapid and effective diagnosis of various diseases. In this study, liquid coplanar-gate graphene field-effect transistors (GFETs) were employed to detect and further distinguish between single-stranded (SS) and double-stranded (DS) DNA molecules. Use of coplanar-gate structure simplified the fabrication steps for GFETs. The liquid coplanar-gate GFETs exhibited higher sensitivity for DNA detection compared to conventional bottom-gate GFETs because they have liquid dielectric layer that was preferred by aqueous DNA. The immobilization of 1-pyrenebutanoic acid succinimidyl ester (PASE) onto graphene surface via π-π interaction further enhanced the DNA sensing performances of GFETs. The base parts of the SS DNA molecules can be covalently linked to the succinimidyl ester group in PASE/graphene, thereby leading to n-doping of graphene by action of lone-pair electrons from nitrogen atoms in the base parts. On the other hand, the negatively charged phosphate groups of the DS DNA molecules exposed to graphene surface induced p-doping of graphene. Accordingly, it was possible to distinguish the single and double-stranded DNA molecules by electrical signals. The combined use of liquid coplanar-system and surface modification of graphene with PASE could decrease the detection limit of DNA molecules to 1 nM. The liquid coplanar-gate organic/graphene hybrid electronics platform developed here will allow rapid and convenient label-free detection of single and double stranded DNA molecules.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Single and double stranded DNAs were detected by organic/graphene hybrid electronics. </LI> <LI> 1-Pyrenebutanoic acid succinimidyl ester was immobilized onto the graphene surface. </LI> <LI> Detection limit of DNA molecules was decreased to 1 nM. </LI> <LI> The liquid coplanar-gate organic/graphene hybrid electronics platform was developed. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

전계효과 트랜지스터(FETs)를 이용한 전하 검출형 DNA 센서에서 Debye length에 따른 검출 감도

송광섭(Kwang Soup Song) 大韓電子工學會 2011 電子工學會論文誌-SC (System and control) Vol.48 No.2

전계효과 트랜지스터(FETs)를 이용한 전하 검출형 DNA센서는 DNA가 가지고 있는 음전하를 중성화 시키는 양이온의 영향은 매우 중요하다. 본 논문에서는 양이온 농도에 의존하는 Debye length에 관한 연구를 통해 DNA 검출감도를 평가하였다. Debye length는 낮은 농도의 NaCl 용액에서 긴 거리를 유지하며, Debye length가 높은 용액에서 DNA가 가지고 있은 음전하는 게이트 채널에 보다 많은 영향을 미친다. 용액내 NaCl농도가 1 mM인 버퍼 용액에서 상보적 DNA의 hybridization에 의한 전계효과 트랜지스터의 게이전압은 21 ㎷ 시프트 했으며, NaCl 농도가 10 mM인 버퍼 용액에서는 7.2 ㎷, NaCl농도가 100mM인 버퍼 용액에서는 전계효과 트랜지스터의 게이트 전압이 5.1 ㎷ 각각 시프트 하였다. 이러한 결과를 바탕으로 전계효과 트랜지스터를 이용한 전하 검출형 DNA센서의 검출 감도는 Debye length에 의존하는 것을 규명하였다. The effects of cations are very important in field-effect transistors (FETs) type DNA sensors detecting the intrinsic negative charge between single-stranded DNA and double-stranded DNA without labeling, because the intrinsic negative charge of DNA is neutralized by cations in electrolyte solution. We consider the Debye length, which depends on the concentration of cations in solution, to detect DNA hybridization based on the intrinsic negative charge of DNA. The Debye length is longer in buffer solution with a lower concentration of NaCl and the intrinsic negative charge of DNA is more effective on the channel surface in longer Debye length solution. The shifts in the gate voltage by DNA hybridization with complementary target DNA are 21 ㎷ in 1 mM NaCl buffer solution, 7.2 ㎷ in 10 mM NaCl buffer solution, and 5.1 ㎷ in 100 mM NaCl buffer solution. The sensitivity of FETs to detect DNA hybridization based on charge detection without labeling depends on the Debye length.

DNA를 이용한 형광페이스트 추적 방법

문혜림,정주연,성기민,임시근 한국과학수사학회 2019 과학수사학회지 Vol.13 No.2

In this study, we utilized artificial DNA to increase the variability of the fluorescent paste. For cost-effectiveness, we amplified the synthesized DNA (AmpDNA) and then diluted this DNA and mixed it with the fluorescent paste. We next confirmed DNA detection within this mixture at ratios of 1:100, 1:1,000, 1:10,000, 1:100,000, and 1:1,000,000. To test the stability of the DNA in various environments, the DNA and fluorescent paste mixtures were mixed at a ratio of 1:10,000 and then incubated under six different environmental conditions. Specifically, these mixtures were applied to an externally exposed pipe and brick, transferred into tubes, and placed at 4 ℃, at 56 ℃ and under fluctuating temperature conditions (shifted between 4 ℃ for 2 weeks and 56 ℃ for 2 weeks). Room temperature conditions were also tested. After 1 month, 6 months, and 12 months, we collected samples and performed PCR using original primers and nested primers to confirm the presence of DNA. DNA was detected under all conditions up to 12 months. This study suggests that AmpDNA can be successfully used as a method to accurately track the original location of the fluorescent paste. Additionally, nested PCR provides a useful method for rapid, simple, and accurate confirmation. 본 연구에서는 형광페이스트의 다양성을 높이고자 인위적으로 제작된 DNA를 활용하였다. 경제성을 높이기 위해, 합성된 DNA를 증폭하여(AmpDNA) 사용하였고, AmpDNA를 희석하여 형광페이스트에 혼합하였다. 1:100, 1:1,000, 1:10,000, 1:100,000 및 1:1,000,000의 비율로 혼합된 혼합물로부터 DNA가 검출됨을 확인하였다. DNA의 안정성 시험을 위해 DNA와 형광페이스트가 1:10,000으로 희석된 혼합물을 6가지 다양한 환경조건(파이프와 벽돌에 도포하여 외부에 노출, 튜브에 옮긴 후 4 ℃, 56 ℃, 4 ℃ 2주 및 56 ℃ 2주 변경 반복, 실온)에서 보관하였다. 1개월, 6개월 및 12개월 후 시료를 채취하였고, original 프라이머와 nested 프라이머를 이용하여 PCR을 수행하였다. 위 6가지 조건으로 12개월 간 보관된 시료들로부터 DNA가 확인되었다. 본 연구결과를 통해 형광페이스트가 묻은 위치를 정확하게 추적하는 방법으로 AmpDNA가 활용될 수 있고, DNA를 검증하는 방법으로 Nested PCR은 신속하고, 간단하면서 정확한 방법으로써 유용하게 사용될 수 있을 것으로 제안한다.

Prevotella nigrescens ATCC $33563^T$ 균주-특이 중합효소연쇄반응 프라이머 개발

송수근,유소영,김미광,김화숙,임선아,김도경,박재윤,국중기,Song, Soo-Keun,Yoo, So-Young,Kim, Mi-Kwang,Kim, Hwa-Sook,Lim, Sun-A,Kim, Do-Kyung,Park, Jae-Yoon,Kook, Joong-Ki 한국미생물학회 2008 미생물학회지 Vol.44 No.3

A Pn10 DNA probe was introduced as a Prevotella nigrescens ATCC $33563^T$-specific DNA probe. In that study, the specificity of the Pn10 was tested with only type or reference strains of 5 oral bacterial species. The purpose of this study is to evaluate the specificity of the Pn10 using the wild type strains of P. nigrescens and is to develop the P. nigrescens ATCC $33563^T$-specific PCR primers based on the nucleotide sequence of the Pn10. The specificity of the Pn10 DNA probe was determined by Southern blot analysis. The nucleotide sequence of Pn10 DNA probes was determined by chain termination method. The PCR primers were designed based on the nucleotide sequence of cloned DNA fragment. The data showed that Pn10 DNA probe were hybridized with the genomic DNAs from P. nigrescens ATCC $33563^T$ and KB6. The Pn10 homologous region, KB6-Pn10, of P. nigrescens KB6 was cloned by PCR and sequenced. The Pn10 and KB6-Pn10 DNA fragments were consisted of 1,875 bp and 1,873 bp, respectively. The percent identity of the two was 98.8% and the divergence of them was 0.6%. The two primer sets (Pn10-F-AC/ Pn10-R-AC and Pn10-F-A/ Pn10-R-A), designed base on the nucleotide sequences of Pn10 DNA probe, were specific to the P. nigrescens ATCC $33563^T$. The two PCR primer sets could detect as little as 4 pg of genomic DNA of P. nigrescens ATCC $33563^T$. These results indicate that the two PCR primer sets have proven useful for the identification of P. nigrescens ATCC $33563^T$, especially with regard to the maintenance of the strain. 본 연구는 Prevotella nigrescens ATCC $33563^T$에 대한 균주 특이 DNA 프로브라고 보고된 Pn10 프로브의 균주 특이성을 한국인에서 분리된 P. nigrescens의 임상분리 균주를 이용하여 검증하고, P. nigrescens ATCC $33563^T$ 균주 특이 PCR 프라이머를 개발하고자 시행되었다. P. nigrescens와 유전학적으로 가장 가까운 Prevotella intermedia를 포함한 구강 내 치주질환 원인균종인 5균종의 표준균주 및 참고균주, 그리고 P. nigrescens와 P. intermedia의 임상분리 균주를 이용하여 Southern blot 분석법을 시행하였다. Southern blot 분석 결과 Pn10 DNA 프로브에 P. nigrescens ATCC $33563^T$ 및 ChDC KB6 두 균주 지놈 DNA가 검출되었다. P. nigrescens KB6 균주에서 Pn10 DNA 프로브와 상동성이 있는 부위를 PCR법으로 증폭(KB6-Pn10)하여 클로닝한 다음 Pn10 DNA프로브와 같이 핵산 염기서열을 결정하여 상동성을 비교하였다. 그 결과 Pn10과 KB6-Pn10의 핵산염기서열간의 Percent identity는 98.8%였으며, divergence는 0.6%였다. Pn10 DNA 프로브의 핵산염기서열을 바탕으로 두 중류 프라이머 쌍(Pn10-F-AC/Pn10-R-AC 및 Pn10-F-A/Pn10-R-A)을 설계 및 제작하여 P. nigrescens ATCC $33563^T$에 대한 균주 특이성을 PCR법으로 검증하였다. 이들 프라이머 쌍들의 민감도(sensitivity) 조사 결과, 이들은 P. nigrescens ATCC $33563^T$ 지놈 DNA 4 pg까지 검출할 수 있음을 알았다. 이상의 연구 결과를 종합하면, Pn10 DNA 핵산염기서열을 바탕으로 설계된 Pn10-F-AC/Pn10-R-AC 및 Pn10-F-A/Pn10-R-A 프라이머 쌍들은 P. nigrescens ATCC $33563^T$를 신속 정확하게 검출하는 수 있어, 균주의 보존적 측면에서 유용하게 이용될 수 있을 것으로 생각된다.

식품으로부터 식중독 세균 검출을 위한 Real-time PCR에 적합한 DNA 추출 방법 비교

구은정(Eun-Jeong Koo),김동호(Dongho Kim),오세욱(Se-Wook Oh) 한국식품과학회 2016 한국식품과학회지 Vol.48 No.4

본 연구에서는 다양한 식품으로부터 식중독 세균의 DNA를 추출하는 효율을 비교 하였다. 사용된 DNA 추출 방법은 TaKaRa Kit를 이용하는 방법, PrepMan reagent를 이용하는 방법, boiling method, PEG를 이용한 alkaline method가 사용되었다. 비용절감이나 시간절약 면에서 boiling method나 PrepMan method도 고려할 수 있지만, column kit를 이용하는 TaKaRa kit가 효율적이라고 판단되었다. 또한 정성 시험법에서 적은 양의 균을 검출하기 위해 증균배양을 거치게 되는데 이때 사용되는 증균배지의 성분이 DNA 추출 후에도 잔류하여 saline과 비교하였을 때 DNA 추출효율이 낮은 결과를 나타내었다. 따라서 증균배지의 성분을 제거한 뒤 DNA를 추출하는 것이 PCR의 효율을 높일 수 있을 것으로 예상된다. 정량 시험법에서는 증균과정을 거치지 않아 균을 검출할 때 DNA의 추출 효율이 중요하기 때문에 DNA 추출 효율을 높이기 위한 가장 좋은 버퍼를 선정하고자 하였다. 그 결과 E. coli O157:H7에서는 saline이, S. aureus에서는 증류수를 버퍼로 사용했을 때 DNA 추출 효율이 가장 높은 것으로 나타났다. This study was conducted to find out the most suitable DNA isolation methods for PCR detection of foodborne pathogens. Four DNA isolation methods including Universal Genomic DNA Extraction Kit (TaKaRa), PrepMan Ultra (Applied Biosystems), boiling method and alkaline lysis method (w/PEG) were tested and compared. The Universal Genomic DNA Extraction kit (TaKaRa) was considered as the more efficient isolation method for Escherichia coli O157:H7 and Staphylococcus aureus in lettuce, fish and beef. Meanwhile to detect the foodborne pathogens directly from foods without enrichment, the four different buffers such as double-distilled water, saline, glycine-saline, glycine-saline with Tween-20 and beef extract were also evaluated. As a result, saline was more suitable buffer for E. coli O157:H7. And double-distilled water was more suitable buffer than saline for S. aureus, respectively

Label-free and high-sensitive detection of Kirsten rat sarcoma viral oncogene homolog and epidermal growth factor receptor mutation using Kelvin probe force microscopy

Jang, K.,Choi, J.,Park, C.,Na, S. Elsevier Applied Science 2017 Biosensors & Bioelectronics Vol.87 No.-

Assessment of Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR) mutations are essential for targeted therapies of patients with non-small-cell lung cancer. In this report, we propose a label-free and high-sensitive detection method of KRAS and EGFR mutations using KPFM and a gold nanoparticle (AuNP)-based platform that densely adsorbs probe DNA and minimizes the sensing area. The detection is based on the evaluation of the surface potential of each AuNP. When AuNPs are modified with probe DNA (AuNP-pDNA), the surface potential is shifted towards the negative potential due to the negatively charged DNA backbone. When AuNP-pDNA further captures target mutant DNA through DNA hybridization, an additional surface potential shift occurs. The platform is able to detect KRAS mutant DNA (13 mer) and EGFR mutant DNA (84 mer) with a limit of detection (LOD) of 3.3pM. Furthermore, the platform is able to detect selectively the KRAS mutant DNA from its wild-type DNA. Our proposed label-free and high-sensitive KPFM method has shown potential glimpses of a personalized medical diagnosis for cancer patients.

Feasibility of quantifying <i>SDC2</i> methylation in stool DNA for early detection of colorectal cancer

Oh, Tae Jeong,Oh, Hyun Il,Seo, Yang Yei,Jeong, Dongjun,Kim, Changjin,Kang, Hyoun Woo,Han, Yoon Dae,Chung, Hyun Cheol,Kim, Nam Kyu,An, Sungwhan BioMed Central 2017 CLINICAL EPIGENETICS Vol.9 No.1

<P><B>Background</B></P><P>Colorectal cancer (CRC) screening is the most efficient strategy to reduce disease-related mortality. Frequent aberrant DNA methylation is known to occur in selected genes and early during CRC development, which has emerged as a new epigenetic biomarker for early detection of CRC. Previously, we reported that we identified that CpG sites of <I>SDC2</I> were aberrantly methylated in tumor tissues of most CRC patients through comprehensive methylation analysis and demonstrated a high potential of quantification of <I>SDC2</I> methylation in blood for early detection of colorectal cancer. In this study, we aim to investigate the feasibility of quantifying <I>SDC2</I> methylation in stool DNA for the early detection of CRC. The objective of this study was to confirm a high frequency of <I>SDC2</I> methylation in tumor tissues at various stages of CRC and investigate the feasibility of a quantitative test for <I>SDC2</I> methylation in fecal DNA by highly sensitive and accurate real-time PCR for early detection of CRC.</P><P><B>Methods</B></P><P>Bisulfite-pyrosequencing assay was performed to measure the <I>SDC2</I> methylation status in tissue samples. For methylation analysis in stool DNA, a highly sensitive and accurate method was applied which implements consecutive two rounds of PCR consisting of unidirectional linear target enrichment (LTE) of <I>SDC2</I> and quantitative methylation-specific real time PCR (qMSP) for <I>SDC2</I>, named as me<I>SDC2</I> LTE-qMSP assay. Its limit of detection was 0.1% methylation (corresponding to ~ 6 copies in total ~ 6200 genome copies).</P><P><B>Results</B></P><P>Positive <I>SDC2</I> methylation was observed in 100% of primary tumors, 90.6% of adenomatous polyps, 94.1% of hyperplastic polyps, and 0% of normal tissues. <I>SDC2</I> methylation level also significantly (<I>P</I> < 0.01) increased according to the severity of lesions. In stool DNA test for <I>SDC2</I> methylation by LTE-qMSP comparing CRC patients with various stages (I to IV) (<I>n</I> = 50) and precancerous lesions (<I>n</I> = 21) with healthy subjects (<I>n</I> = 22), the overall sensitivity was 90.0% for detecting CRC and 33.3% for detecting small polyps, with a specificity of 90.9%.</P><P><B>Conclusions</B></P><P>Taken together, our result indicates that stool DNA-based <I>SDC2</I> methylation test by LTE-qMSP is a potential noninvasive diagnostic tool for early detection of CRC.</P>

SERS-based genetic assay for amplification-free detection of prostate cancer specific PCA3 mimic DNA

Yu, Jimin,Jeon, Jinhyeok,Choi, Namhyun,Lee, Jin Oh,Kim, Young-Pil,Choo, Jaebum Elsevier 2017 Sensors and actuators. B, Chemical Vol.251 No.-

<P><B>Abstract</B></P> <P>We report the development of amplification-free surface-enhanced Raman scattering (SERS)-based DNA assays for the rapid and highly sensitive detection of prostate cancer antigen 3 (PCA3) mimic DNA. This technique does not require any DNA amplification process using thermo-cycles in conventional polymerase chain reaction (PCR) due to its highly sensitive detection capability. Herein, the PCA3 mimic DNA, composed of 45 nucleotide sequences, was sandwiched between two probe DNA-immobilized particles (ASO<SUB>735</SUB>-conjugated detection HGNs and ASO<SUB>683</SUB>-immobilized capture magnetic beads) by hybridization reactions. Its quantitative analysis was performed by monitoring the characteristic Raman peak intensity of sandwich DNA complexes. The limit of detection (LOD) is estimated to be 2.7fM, which is about four orders of magnitude more sensitive than that of conventional PCR. This SERS-based DNA assay technique is expected to be a potentially useful tool for early disease diagnosis.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We developed a SERS-based biosensor for the highly sensitive detection of PCA3 mimic DNA. </LI> <LI> DNA amplification process is not required for this SERS-based assay platform. </LI> <LI> This method has strong potential to be a tool for early genetic disease diagnosis. </LI> </UL> </P>