Ye Lim Jung

Direct colorimetric diagnosis of pathogen infections by utilizing thiol-labeled PCR primers and unmodified gold nanoparticles

We describe here a greatly simplified colorimetric detection method to identify PCR-amplified nucleic acids. Our method relies on the PCR product having thiol group at one end, which is generated by employing thiolated PCR primer. After PCR amplification reaction, unmodified gold nanoparticles (AuNPs) are added into the reaction tube followed by the addition of NaCl to induce the aggregation of AuNPs. The PCR products strongly bind to the surface of AuNPs through the interaction of the terminal thiol groups and the long chain of DNA which has abundant negative charges enhances the electrostatic and steric repulsion among AuNPs, which consequently leads to the prevention of the salt-induced aggregation. As a result, the color of AuNPs remains red in the presence of the PCR-amplified nucleic acids, while the AuNPs change its color from red to blue due to the salt-induced aggregation in the absence of the PCR products. This simple but very efficient colorimetric strategy was successfully demonstrated by diagnosing Chlamydia infection using a real human urine sample. Since the results can be clearly seen with the naked eye without any complicated step such as surface modification of AuNPs or PCR product purification, this method can be easily applied to point-of-care diagnosis.

Investigation of the signaling mechanism and verification of the performance of an electrochemical real-time PCR system based on the interaction of methylene blue with DNA

The operation of an electrochemical real-time PCR system, based on intercalative binding of methylene blue (MB) with dsDNA, has been demonstrated. PCR was performed on a fabricated electrode-patterned glass chip containing MB while recording the cathodic current peak by measuring the square wave voltammogram (SWV). The current peak signal was found to decrease with an increase in the PCR cycle number. This phenomenon was found to be mainly a consequence of the lower apparent diffusion rate of the MB-DNA complex (D(b) = 6.82 × 10(-6) cm(2) s(-1) with 612 bp dsDNA) as compared to that of free MB (D(f) = 5.06 × 10(-5) cm(2) s(-1)). Utilizing this signal changing mechanism, we successfully demonstrated the feasibility of an electrochemical real-time PCR system by accurately quantifying initial copy numbers of Chlamydia trachomatis DNA templates on a direct electrode chip. A standard calibration plot of the threshold cycle (C(t)) value versus the log of the input template quantity demonstrated reliable linearity and a good PCR efficiency (106%) that is comparable to that of a conventional TaqMan probe-based real time PCR. Finally, the system developed in this effort can be employed as a key technology for the achievement of point-of-care genetic diagnosis based on the electrochemical real-time PCR.

Colorimetric SNP Genotyping Based on Allele-Specific PCR by Using a Thiol- Labeled Primer

chemistry. [7] However, these methods usually require multiple manipulations, high levels of technical expertise, and expensive instruments, all of which makes them unsuitable for pointof-care testing (POCT) applications. Consequently, a great incentive exists for the development of simple, rapid, and costeffective SNP genotyping methods that are suitable for POCT in facility-limited environments. Colorimetric methods fulfill most of the above requirements as they enable cost-effective and on-site detection without the need for sophisticated equipment. To date, a number of such methods for SNP detection that utilize, for example, metal nanoparticles, [8] peroxidase-mimicking DNAzymes, [9] and peptide nucleic acids (PNAs), [1] have been reported. Among these, gold nanoparticle (AuNP)-based approaches [10–12] have received great attention owing to their unique optical properties, robustness, and high surface areas, which make them ideally suited as signaling probes in colorimetric-detection platforms. [12] However, the requirement for modification of the AuNP surface with oligonucleotide probes and/or the need for precise temperature control during the assay significantly limits applications of AuNP-based methods. In recent studies, we developed a novel strategy based on unmodified AuNPs for the colorimetric detection of target nucleic acids that are amplified by thiol-labeled primers without time-consuming and complicated procedures. [13] We successfully demonstrated its diagnostic utility by reliably identifying Chlamydia trachomatis infection. Here, we have extended our previous strategy for nucleic acid detection by devising a novel method for SNP identification that incorporates a modified allele-specific PCR (ASPCR) method. In principle, ASPCR is a cost-effective procedure that characterizes SNPs based on differences between the PCR efficiency of allele-specific primers. [14] The primers consist of sequences adjacent to the polymorphic site that are complementary to the allelic variant and differ only in the terminal nucleotide at the 3’-end. [2] In the first step of our new assay procedure, ASPCR is performed with a thiolated primer. In the second step, AuNPs are mixed with the products and then subjected to salt-induced aggregation. The resulting color of the solution (i.e., red or blue) indicates the genotype at the SNP site because only the thiolated PCR products provide the AuNPs with a significant resistance to salt-induced aggregation and inhibit the red-to-blue color transition. The strategy we have devised for SNP identification is schematically illustrated in Figure 1 for the detection of a single base mutation. In the first step, ASPCR is performed separately in four different reaction tubes containing each of the four thiolated forward primers, which differ only in the terminal base at their 3’-ends (A, T, C and G). When the base at the 3’end of the forward primer is complementary to the antisense strand of the genomic target DNA, PCR amplification occurs and generates PCR amplicons labeled with a thiol group at the 5’-end of one strand. In contrast, the other three primers, which have a noncomplementary nucleotide at their 3’-ends, cannot be extended and thus they fail to generate PCR amplicons. The existence of the thiol-labeled PCR products is then colorimetrically determined by sequentially adding unmodified AuNPs and NaCl to each tube. When thiolated PCR amplicons are mixed with AuNPs, they bind to the surface of unmodified AuNPs through a strong gold–thiol interaction. The electrostatic repulsion between the AuNPs is enhanced as a consequence of the large number of negative charges on their surfaces due to the negatively charged phosphate backbone of the bound DNA. In addition, steric repulsion between the AuNPs is increased because the longer (compared to the diameter of AuNPs) DNA grafted onto the AuNPs surface forms a thick polymeric barrier that prevents the particles from approaching each other. [15] As a consequence of these “electrostatic/steric stabilization effects”, [16] salt-induced aggregation of AuNPs is significantly inhibited, and the solution remains red when salt is added. In contrast, in the absence of the thiol-labeled PCR amplicon, AuNPs undergo immediate aggregation upon salt addition; this results in a colorimetric transition from red to blue. By employing this strategy, the genotypes at mutation sites can be conveniently determined based on the color of the sample. To prove the conceptual basis of this novel strategy for SNP genotyping and to confirm the crucial role played by thiolated primers in the AuNPs-based colorimetric assay, ASPCR was per

Label-free colorimetric detection of biological thiols based on target-triggered inhibition of photoinduced formation of AuNPs.

A label-free colorimetric method for the detection of biological thiols (biothiols) was developed. This method is based on prevention of the photoinduced reduction of auric ions (Au(III)) in the presence of amino acids (acting as a reducing agent) by biothiols; the photoinduced reduction is inhibited due to the strong interaction of the biothiols with Au(III). In this method, the sample was first incubated in an assay solution containing Au(III) and threonine; the sample solution was then exposed to 254 nm UV light. For samples without biothiols, this process led to the photoreduction of Au(III) followed by growth of gold nanoparticles accompanied by the visually detectable development of a red coloration typified by an absorption peak at ca 530 nm. Conversely, in the presence of biothiols, reduction of Au(III) to Au(0) was prevented by entrapment of Au(III) within the biothiols via the thiol group. The solution thus remained colorless even after UV irradiation, which was used as an indicator of the presence of biothiols. Using this strategy, biothiols were very conveniently analyzed by monitoring color changes of the samples with the naked eye or a UV-vis spectrometer. The strategy based on this interesting phenomenon exhibited high selectivity toward biothiols over common amino acids and was successfully employed for reliable quantification of biothiols present in human plasma, demonstrating its great potential for clinical applications.

A Sexually Transmitted Disease (STD) DNA chip for the diagnosis of genitourinary infections

The results of an investigation aimed at the development of a DNA chip for the detection of genitourinary infections are described. Through analysis of over 35,000 clinical cases, 14 pathogens which are most abundantly found among Koreans were selected and candidate sequences for capture probes were accordingly chosen by considering their sequences and β-globin house-keeping gene. Among this group, the most suitable capture probe sequences were selected by employing repeated chip tests in which they are immobilized on a glass chip by using a recently developed novel gold nanoparticles-based method. A multiplex PCR method was established to generate fluorescence-labeled sequences for all 14 pathogens along with the β-globin gene. By using optimized hybridization conditions, the final chip was constructed and employed to diagnose reliably both single and multiple infections in clinical human samples for 14 target pathogens. The results show that the novel chip methodology serves as a highly reliable and convenient tool for the diagnosis of Sexually Transmitted Diseases (STDs). Furthermore, this study has its great significance in that it demonstrates the entire process from statistical analysis of a large number of clinical cases to the final development of STD DNA chip just ready to be applied or commercialized in the clinical diagnostic field.

A mass spectrometry-based multiplex SNP genotyping by utilizing allele-specific ligation and strand displacement amplification

We herein describe a new mass spectrometry-based method for multiplex SNP genotyping by utilizing allele-specific ligation and strand displacement amplification (SDA) reaction. In this method, allele-specific ligation is first performed to discriminate base sequence variations at the SNP site within the PCR-amplified target DNA. The primary ligation probe is extended by a universal primer annealing site while the secondary ligation probe has base sequences as an overhang with a nicking enzyme recognition site and complementary mass marker sequence. The ligation probe pairs are ligated by DNA ligase only at specific allele in the target DNA and the resulting ligated product serves as a template to promote the SDA reaction using a universal primer. This process isothermally amplifies short DNA fragments, called mass markers, to be analyzed by mass spectrometry. By varying the sizes of the mass markers, we successfully demonstrated the multiplex SNP genotyping capability of this method by reliably identifying several BRCA mutations in a multiplex manner with mass spectrometry.

Direct detection of unamplified genomic DNA based on photo-induced silver ion reduction by DNA molecules

A label-free, colorimetric method has been developed for ultrasensitive detection of nucleic acids that is based on photoinduced silver ion (Ag(+)) reduction around DNA bases. The assay system is capable of directly detecting bacterial genomic DNA without the need for PCR amplification.

Gold Nanoparticles - based Colorimetric Single Nucleotide Polymorphisms Genotyping Utilizing Allele-specific PCR

A very convenient colorimetric method for the identification of single nucleotide polymorphism was developed utilizing gold nanoparticles and an allele-specific polymerase chain reaction and its diagnostic capability was successfully verified by correctly identifying various mutations in the BRCA1 gene.