Session: 13-04-01: Applications of Micro and Nano Systems in Medicine and Biology

Paper Number: 147275

147275 - Laser-Induced Graphene Heater for Dna Amplification and Crispr-Cas9 Biosensing 

Laser-Induced Graphene (LIG) was pursued as a low-power heater for isothermal Loop-Mediated Isothermal Amplification (LAMP) DNA amplification, achieving rapid amplification within 30 minutes with only 200 mW. Compared to traditional thermal cyclers, LIG heaters offer substantial power reduction, cost efficiency, and simplified use. Our method streamlines the DNA amplification and detection, representing an efficient and sensitive approach for detecting nucleic acids. Simultaneously, a cost-effective CRISPR-Cas9 sensor was created that detects DNA concentrations as low as 16 pM.

LIG was engineered on a polyimide tape utilizing a precise laser-induced carbonization technique that employs laser irradiation to selectively transform the polyimide tape into a patterned graphene structure. The resulting LIG is securely attached as a heating element onto one surface of a glass substrate of 100 μm thickness. On the opposite side of the glass, was a microchannel glass layer with inlet and outlet ports, and a spacer of UV-cured adhesive 200μm thick. The LAMP enzyme and primers along with the target DNA were injected into the microchannel, and a voltage was applied across the LIG heater to achieve the optimal 65 °C LAMP amplification temperature that was maintained within the microchannel. Agarose gel electrophoresis and lateral flow assays (LFA) were employed to detect the amplification products that were compared to DNA amplified using a thermocycler instrument which has a power consumption higher than 100 W.

Lateral flow assays (LFA), such as that used for at home pregnancy or COVID testing, enable detection of target analytes. In the experiment, agarose gel electrophoresis and LFA results were analyzed to observe DNA amplicons produced by the LAMP using LIG heater and thermal cycling across different incubation durations. Both methodologies successfully amplified DNA within a 30-minute timeframe, exhibiting discernible positive signals in LFA tests.

DNA can also be detected by an electrochemical sensor and, using our pyrolytic glassy carbon electrodes, we constructed a low cost CRISPR-Cas9-based detection device for specific bonding with target DNA to achieve selective detection. The process of sensor electrodes are initially immersed for 20 minutes in a solution containing the target DNA. Subsequently, 10 mM Hexaammineruthenium(III) chloride (RuHex) is deposited onto the electrode surface, followed by five-minutes of incubation. The oxidation peak of RuHex is then detected in the Tris-EDTA buffer using Differential Pulse Voltammetry (DPV). The electrode surface is rinsed with nuclease-free water before each procedural step. The response and calibration curves of the CRISPR Cas9 biosensor to varying concentrations of target DNA are presented, demonstrating increased DNA capture on the electrode surface by CRISPR-Cas9 with rising DNA concentration . Since RuHex can bind with double-stranded DNA, more RuHex is attached to the electrode surface, resulting in a higher oxidation peak during the DPV process. Under these conditions, the lowest concentration tested was 10,000 copies/µL, equivalent to 16 pM of target DNA.

In conclusion, the integration of Laser-Induced Graphene as a low-power heater and a CRISPR-Cas9 sensor represents a powerful approach to DNA amplification and detection. This innovative combination offers substantial advantages in terms of power efficiency, cost-effectiveness, and workflow compared to traditional methods and highlights the potential for point of collection applications. The developed electrochemical sensor further enhances specificity and selectivity in DNA detection. Overall, this study represents a resource-efficient approach for DNA amplification and detection.

Presenting Author: Peng Zhou University of Minnesota

Presenting Author Biography: Peng Zhou was born in Ningguo, Anhui, China in 1993. He received the B.S. degree from the Department of Mechanical Engineering, Beijing Institute of Technology, China, in 2015, and the M.S. degree in mechanical engineering from Purdue University Northwest, USA. He received his Ph.D. degree from the Department of Mechanical Engineering, University of Minnesota, in 2023. He is currently a postdoctoral researcher in the Department of Mechanical Engineering, University of Minnesota.
His research interests include the photocatalytic water purification and MEMS-based microsensors for both environmental and biomedical applications.

Authors:

Peng Zhou University of Minnesota
Amber Mcelroy University of Minnesota
Yingming Xu University of Minnesota
Terrence Simon University of Minnesota
Mark Osborn University of Minnesota
Tianhong Cui University of Minnesota