Detection of Deoxyribonucleic Acid (DNA) Targets Using Polymerase Chain Reaction (PCR) and Paper Surface-Enhanced Raman Spectroscopy (SERS) Chromatography

Abstract

Surface-enhanced Raman spectroscopy (SERS) enables multiplex detection of analytes using simple, portable equipment consisting of a single excitation source and detector. Thus, in theory, SERS is ideally suited to replace fluorescence in assays that screen for numerous deoxyribonucleic acid (DNA) targets, but in practice, SERS-based assays have suffered from complexity and elaborate processing steps. Here, we report an assay in which a simple inkjet-fabricated plasmonic paper device enables SERS-based detection of multiple DNA targets within a single polymerase chain reaction (PCR). In prior work, we demonstrated the principles of chromatographic separation and SERS-based detection on inkjet-fabricated plasmonic paper. The present work extends that capability for post-PCR gene sequence detection. In this design, hydrolysis DNA probes with 5' Raman labels are utilized; if the target is present, the probe is hydrolyzed during PCR, freeing the reporter. After applying the PCR sample to a paper SERS device, an on-device chromatographic separation and concentration is conducted to discriminate between hydrolyzed and intact probes. SERS is then used to detect the reporter released by the hydrolyzed probes. This simple separation and detection on paper eliminates the need for complex sample processing steps. In this work, we simultaneously detect the methicillin-resistant Staphylococcus aureus genes mecA and femB to illustrate the concept. We envision that this approach could contribute to the development of multiplex DNA diagnostic tests enabling screening for several target sequences within a single reaction, which is necessary for cases in which sample volume and resources are limited.

Review of Surface Enhanced Raman Scattering Applications in Forensic Science

“…Spectrosc. 2014 , 68 , 909–915 (ref ). Copyright 2014 with permission of Society for Applied Spectroscopy.…”

Surface enhanced Raman scattering for the multiplexed detection of pathogenic microorganisms: towards point-of-use applications

“…In this study, MRSA genes MecA and FemA, which are used to identify S. aureus and methicillin resistance, were modified with Raman reporters and these samples were loaded into a PCR well separated from an upper well containing silver colloid by the dialysis membrane. 116 The microchips were then sealed and placed in a custom thermocycler in which thermal cycles and spectrometer readings were synchronised. SERS data was collected at the end of every reaction cycle and signal intensity was measured at the height of Fig.…”

Surface enhanced Raman spectroscopy (SERS) for in vitro diagnostic testing at the point of care

“…The additional push for one technology to monitor several biomarkers simultaneously for more effective and universal disease diagnosis is motivating recent advancements in multiplexed sensing capabilities. Many optical approaches have been utilized for POC applications including fluorescence [69][70][71][72], luminescence, absorbance, Forster energy transfer (FRET) [73], bioluminescence energy transfer (BRET) [74][75][76], surfaceplasmon resonance (SPR) [77,78], resonant Rayleigh scattering [79][80][81][82], geometric scattering [1], and Raman spectroscopic approaches [83][84][85][86][87][88][89]. Advancements in biochemical sensing methods, nanotechnology, and the miniaturization of optics have been key in improving spectroscopic platforms for biomarker detection.…”