Image created by Dr. Michael J. Miller
PCR genetic analysis has been in the spotlight since COVID-19, but light is now further facilitating PCR-free methods.
Osaka Metropolitan University scientists have developed a light-induced DNA detection technique, using heterogeneous probe particles, that enables ultra-sensitive and ultra-fast genetic analysis without the need for PCR amplification. This advancement is lighting the way for faster, more affordable, and precise genetic analysis across medicine, environmental science, and portable diagnostics.
As a means of analyzing changes in DNA, genetic testing — which is essential for diagnosing infectious diseases, detecting early-stage cancer, verifying food safety, and analyzing environmental DNA — has long relied on PCR (polymerase chain reaction) as the gold standard. Since the COVID-19 pandemic, the term “PCR” has become part of our common vocabulary. But as those of us who have experienced them know, PCR tests are neither cheap nor fast; they typically require centralized labs, bulky equipment, and specially trained personnel.
“Our light-induced method detects DNA without the need for PCR,” write Shuichi Toyouchi, a Project Lecturer, Prof. Shiho Tokonami, the Deputy Director, and Takuya Iida, the Director at Osaka Metropolitan University’s Research Institute for Light-induced Acceleration System (RILACS), as the lead authors of this study.
Unlike PCR, which amplifies DNA sequences by making millions of copies of target DNA for detection, this method directly detects DNA by concentrating it and enhancing specificity through strong optical forces and photothermal effect.
The team developed a system using heterogeneous probe particles, including gold nanoparticles and polystyrene microparticles. These probes are short, known DNA sequences designed to hybridize, or bind, with complementary sequences in the target DNA. This process, known as DNA hybridization, allows the matching strands to bind together, making the pairing detectable through fluorescence.
The researchers then irradiated, with laser light, the solution containing the target DNA and probe particles. When the particle size matches the laser wavelength, a phenomenon called Mie scattering occurs, generating optical forces that move the particles and accelerate DNA hybridization. The gold nanoparticles absorb laser light, creating localized heat, or photothermal effect, to further enhance the hybridization specificity.
“Using just about five minutes of laser light irradiation, our method demonstrated great potential for accurate mutation detection with a sensitivity one order of magnitude higher than that of digital PCR.,” Toyouchi, Tokonami, and Iida write.
By eliminating the need for PCR amplification, this method reduces costs, simplifies testing, and accelerates results, making genetic analysis more accessible in daily life applications — from healthcare and food safety to environmental conservation and personal health tracking.
“We aim to apply this PCR-free technology to high-sensitivity cancer diagnostics, quantum life science research, and even at-home or environmental DNA testing,” Iida said.
The study was published in ACS Sensors.
Funding
JST Mirai Program (No. JPMJMI18GA, No. JPMJMI21G1),
Grant-in-Aid for Scientific Research (A) (No. JP17H00856, No. JP21H04964, No. JP24H00433),
JST FOREST Program (No. JPMJFR201O),
Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas (No. JP16H06507), Grant-in-Aid for Research Activity Start-up (No. JP22K20512 and No. JP24K23034), Grant-in-Aid for Early-Career Scientists (No. 20K15196), Grant-in-Aid for JSPS Research Fellows (No. 21J21304), Grant-in-Aid for Scientific Research (C) (No. JP24K08282), Grant-in-Aid for Transformative Research Areas (A) (No. JP23H04594),
Key Project Grant Program of Osaka Prefecture University.
Reference:
Journal: ACS Sensors
Title: Single Nucleotide Polymorphism Highlighted via Heterogeneous Light-Induced Dissipative Structure
DOI: 10.1021/acssensors.4c02119
Author: Shuichi Toyouchi, Seiya Oomachi, Ryoma Hasegawa, Kota Hayashi, Yumiko Takagi, Mamoru Tamura, Shiho Tokonami, and Takuya Iida
Published: 23 January 2025
URL: https://doi.org/10.1021/acssensors.4c02119
Abstract
The unique characteristics of biological structures depend on the behavior of DNA sequences confined in a microscale cell under environmental fluctuations and dissipation. Here, we report a prominent difference in fluorescence from dye-modified single-stranded DNA in a light-induced assembly of DNA-functionalized heterogeneous probe particles in a microwell of several microliters in volume. Strong optical forces from the Mie scattering of microparticles accelerated hybridization, and the photothermal effect from the localized surface plasmons in gold nanoparticles enhanced specificity to reduce the fluorescence intensity of dye-modified DNA to a few %, even in a one-base mismatched sequence, enabling us to clearly highlight the single nucleotide polymorphisms in DNA. Fluorescence intensity was positively correlated with complementary DNA concentrations ranging in several tens fg/μL after only 5 min of laser irradiation. Remarkably, a total amount of DNA in an optically assembled structure of heterogeneous probe particles was estimated between 2.36 ymol (2.36 × 10–24 mol) and 2.36 amol (2.36 × 10–18 mol) in the observed concentration range. These findings can promote an innovative production method of nanocomposite structures via biological molecules and biological sensing with simple strategies avoiding genetic amplification in a PCR-free manner.