Raman spectroscopic analysis of skin as a diagnostic tool for Human African Trypanosomiasis.

Human African Trypanosomiasis (HAT) has been responsible for several deadly epidemics throughout the 20th century, but a renewed commitment to disease control has significantly reduced new cases and motivated a target for the elimination of Trypanosoma brucei gambiense-HAT by 2030. However, the recent identification of latent human infections, and the detection of trypanosomes in extravascular tissues hidden from current diagnostic tools, such as the skin, has added new complexity to identifying infected individuals. New and improved diagnostic tests to detect Trypanosoma brucei infection by interrogating the skin are therefore needed. Recent advances have improved the cost, sensitivity and portability of Raman spectroscopy technology for non-invasive medical diagnostics, making it an attractive tool for gambiense-HAT detection. The aim of this work was to assess and develop a new non-invasive diagnostic method for T. brucei through Raman spectroscopy of the skin. Infections were performed in an established murine disease model using the animal-infective Trypanosoma brucei brucei subspecies. The skin of infected and matched control mice was scrutinized ex vivo using a confocal Raman microscope with 532 nm excitation and in situ at 785 nm excitation with a portable field-compatible instrument. Spectral evaluation and Principal Component Analysis confirmed discrimination of T. brucei-infected from uninfected tissue, and a characterisation of biochemical changes in lipids and proteins in parasite-infected skin indicated by prominent Raman peak intensities was performed. This study is the first to demonstrate the application of Raman spectroscopy for the detection of T. brucei by targeting the skin of the host. The technique has significant potential to discriminate between infected and non-infected tissue and could represent a unique, non-invasive diagnostic tool in the goal for elimination of gambiense-HAT as well as for Animal African Trypanosomiasis (AAT).

Rapid Prototypable Biomimetic Peristalsis Bioreactor Capable of Concurrent Shear and Multi-Axial Strain.

Peristalsis is a nuanced mechanical stimulus comprised of multi-axial strain (radial and axial strain) and shear stress. Forces associated with peristalsis regulate diverse biological functions including digestion, reproductive function, and urine dynamics. Given the central role peristalsis plays in physiology and pathophysiology, we were motivated to design a bioreactor capable of holistically mimicking peristalsis. We engineered a novel rotating screw-drive based design combined with a peristaltic pump, in order to deliver multi-axial strain and concurrent shear stress to a biocompatible polydimethylsiloxane (PDMS) membrane "wall." Radial indentation and rotation of the screw drive against the wall demonstrated multi-axial strain evaluated via finite element modeling. Experimental measurements of strain using piezoelectric strain resistors were in close alignment with model-predicted values (15.9 ± 4.2% vs. 15.2% predicted). Modeling of shear stress on the "wall" indicated a uniform velocity profile and a moderate shear stress of 0.4 Pa. Human mesenchymal stem cells (hMSCs) seeded on the PDMS "wall" and stimulated with peristalsis demonstrated dramatic changes in actin filament alignment, proliferation, and nuclear morphology compared to static controls, perfusion, or strain, indicating that hMSCs sensed and responded to peristalsis uniquely. Lastly, significant differences were observed in gene expression patterns of calponin, caldesmon, smooth muscle actin, and transgelin, corroborating the propensity of hMSCs toward myogenic differentiation in response to peristalsis. Collectively, our data suggest that the peristalsis bioreactor is capable of generating concurrent multi-axial strain and shear stress on a "wall." hMSCs experience peristalsis differently than perfusion or strain, resulting in changes in proliferation, actin fiber organization, smooth muscle actin expression, and genetic markers of differentiation. The peristalsis bioreactor device has broad utility in the study of development and disease in several organ systems.

Paper Microfluidic Device with a Horizontal Motion Valve and a Localized Delay for Automatic Control of a Multistep Assay.

A microfluidic paper-based analytical device (µPAD) is a cost-effective platform to implement assays, especially for point-of-care testing. Developing µPADs with fluidic control is important to implement multistep assays and provide high sensitivities. However, current localized delays in µPADs made of sucrose have a limited ability to decrease the flow rate. In addition, existing µPADs for automatic multistep assays are limited by their need for auxiliary instruments, their false activation, or their unavoidable tradeoff between available fluid volumes and temporal differences between steps. Here, a novel µPAD composed of a localized dissolvable delay and a horizontal motion mechanical valve for use as an automatic multistep assay is reported. A mixture of fructose and sucrose was used in the localized dissolvable delay and it provided an effective decrease in the flow rate to ensure adequate sensitivity in an assay. The dissolvable delay effectively doubled the flow time. A mechanical valve using a horizontal movement was developed to automatically implement a multistep process. Two-step and four-step processes were enabled with the µPAD. Cardiac troponin I (cTnI), a gold-standard biomarker for myocardial infarction, was used as a model analyte to show the performance of the developed µPAD in an assay. The designed µPAD, with the simple-to-make localized dissolvable delay and the robust mechanical valve, provides the potential to automatically implement high-performance multistep assays toward a versatile platform for point-of-care diagnostics.

Detection of cardiovascular disease associated miR-29a using paper-based microfluidics and surface enhanced Raman scattering.

The development of viable point-of-care diagnostic formats is integral to achieving better patient care and improved outcomes. The need for robust and low-cost tests is especially important in under-resourced and rural settings. Perhaps the greatest challenge is ensuring that an untrained individual is capable of operating and interpreting the test, out with a care facility. Here we present a paper-based diagnostic device capable of sensing miR-29a using both colorimetric and surface enhanced Raman scattering (SERS) analysis. Rather, than carry out the two types of analyses in tandem, we envisage that the colorimetric output is easy enough to be interpreted by the untrained-individual administering the test to provide them with qualitative feedback. If deemed positive, the test can be further validated at a centralized care facility using a handheld-Raman spectrometer to provide a semi-quantitative result. Detection of miR-29a, a microRNA associated with myocardial infarction, was achieved at a level of pg µL-1 through the combination of three-dimensional paper-based microfluidics, colorimetric detection, and surface enhanced Raman scattering (SERS) analysis. RGB analysis of the colorimetric output generated from samples containing miR-29a at different concentrations (18-360 pg µL-1) showed differentiation from the control sample, however significant repeat variability indicated that it could not be used for quantifying miR-29a levels. However, the SERS analysis exhibited greater reproducibility at varying concentrations, achieving an LoD of 47 pg µL-1. The union of the paper-based device and the two analysis methods resulted in the production of a sensitive, reproducible and facile, point of care test (POCT), which paves the way for future implementation in the diagnosis of a range of diseases.

A SERS approach for rapid detection of microRNA-17 in the picomolar range.

Epigenetic biomarkers are powerful tools for early disease detection and are particularly useful for elusive conditions like preeclampsia. Predicting preeclampsia at an early stage is one of the most important goals of maternal-fetal medicine. To this end, recent studies have identified microRNAs-such as microRNA-17-as early biomarkers for preeclampsia. Yet clinical applications are lagging, owing in part to the sensing challenges presented by the biomarkers' small size and complex environment. Surface enhanced Raman spectroscopy (SERS) is an emergent optical technique that is recognized for its potential to overcome these challenges. In this study, DNA functionalized nanoparticles were designed as probes to capture and quantify miRNA-17 in solution. SERS was used to determine the presence and concentration of miRNA-17 based on the formation of plasmonic nanoparticle aggregates. The miRNA-17 assay was tested at concentrations of 1 pM to 1 nM in both PBS and a representative complex biological sample. In both situations the assay was unaffected by non-complementary microRNA samples. These results demonstrate SERS's specificity and sensitivity for a new biomarker (miRNA-17) that may ultimately be used in a detection platform for early diagnosis of preeclampsia.

Towards establishing a minimal nanoparticle concentration for applications involving surface enhanced spatially offset resonance Raman spectroscopy (SESORRS) in vivo.

Resonant chalcogenpyrylium nanotags demonstrate an exceptional surface enhanced Raman scattering (SERS) performance for use in SORS applications. Using surface enhanced spatially offset Raman spectroscopy (SESORS), nanotags modified with a chalcogenpyrylium dye were observed at concentrations as low as 1 pM through 5 mm of tissue. Calculated limits of detection suggest that these SERS nanotags can be detected at 104 fM using surface enhanced spatially offset resonance Raman scattering (SESORRS) demonstrating their potential for in vivo applications.

Surface enhanced resonance Raman spectroscopy (SERRS) for probing through plastic and tissue barriers using a handheld spectrometer.

The ability to probe through barriers and tissue non-invasively is an urgent unmet need in both the security and biomedical imaging fields. Surface enhanced Raman spectroscopy (SERS) has been shown to yield superior enhancement in signal over conventional Raman techniques. Furthermore, by utilising a resonant Raman reporter to produce surface enhanced resonance Raman spectroscopy (SERRS), even greater enhancement in chemical signal can be generated. Here we show the benefit of using red-shifted chalcogenpyrylium based Raman reporters for probing through large thicknesses of plastic and tissue barriers using a conventional Raman instrument. In addition, the benefit of using a resonant Raman reporter for superior levels of through barrier detection is demonstrated, and we aim to show the advantage of using resonant nanotags in combination with conventional Raman spectroscopy to probe through plastic and tissue barriers. Raman signals were collected from SERRS active nanotags through plastic thicknesses of up to 20 mm, as well as the detection of the same SERRS nanotags through up to 10 mm of tissue sections using a handheld conventional Raman spectrometer. The ability to detect SERRS-active nanotags taken up into ex vivo tumour models known as multicellular tumour spheroids (MTS), through depths of 5 mm of tissue is also shown. The advantages of applying multivariate analysis for through barrier detection when discriminating analytes with similar spectral features as the barrier is also clearly demonstrated. To the best of our knowledge, this is the first report of the assessment of the maximum level of through barrier detection using a conventional handheld Raman instrument for SERS applications as well as demonstration of the power of resonant nanotags for probing through barriers using conventional Raman spectroscopy.

From synthetic DNA to PCR product: detection of fungal infections using SERS.

We report the use of silver hydroxylamine nanoparticles functionalised with single stranded monothiolated DNA for the detection of fungal infections. The four different species of fungi that were targeted were Candida albicans, Candida glabrata, Candida krusei and Aspergillus fumigatus. Rational design of synthetic targets and probes was carried out by carefully analysing the 2-D folding of the DNA and then by global alignment of the sequences to ensure specificity. The effects of varying the concentrations of the DNA and dye surrounding the nanoparticles on the resultant surface enhanced Raman scattering (SERS) signal were also investigated to ensure compatibility of the probes in a multiplexed environment. Using principal components analysis (PCA) it was possible to detect the individual presence of each target and group them accordingly. The move to detect the C. krusei single stranded PCR product (ssPCR) was significant to demonstrate that the methodology could be employed for the detection and diagnosis of invasive fungal infections (IFDs) within a clinical setting. Initially the PCR product was subjected to an alkali shock method in order to separate the strands ready for detection using the nanoparticle probes system. This time 18 base probes were employed to enhance hybridisation efficiency and dextran sulfate was found to have a vital role in ensuring that detection of the C. krusei target was achieved. This demonstrated the use of DNA functionalised silver nanoparticle for the detection of clinically relevant DNA relating to a specific fungal infection and offers significant promise for future diagnostic applications.

Detection of potentially toxic metals by SERS using salen complexes.

Surfaced enhanced Raman scattering (SERS) can discriminate between metal complexes due to the characteristic "spectral fingerprints" obtained. As a result, SERS has the potential to develop relatively simple and sensitive methods of detecting and quantifying a range of metal ions in solution. This could be beneficial for the environmental monitoring of potentially toxic metals (PTMs). Here, salen was used as a ligand to form complexes of Ni(ii), Cu(ii), Mn(ii) and Co(ii) in solution. The SERS spectra showed characteristic spectral differences specific to each metal complex, thus allowing the identification of each of these metal ions. This method allows a number of metal ions to be detected using the same ligand and an identical preparation procedure. The limit of detection (LOD) was determined for each metal ion, and it was found that Ni(ii), Cu(ii) and Mn(ii) could be detected below the WHO's recommended limits in drinking water at 1, 2 and 2 µg L-1, respectively. Co(ii) was found to have an LOD of 20 µg L-1, however no limit has been set for this ion by the WHO as the concentration of Co(ii) in drinking water is generally <1-2 µg L-1. A contaminated water sample was also analysed where Mn(ii) was detected at a level of 800 µg L-1.

Application of surface enhanced Raman scattering to the solution based detection of a popular legal high, 5,6-methylenedioxy-2-aminoindane (MDAI).

The ever increasing numbers and users of designer drugs means that analytical techniques have to evolve constantly to facilitate their identification and detection. We report that surface enhanced Raman scattering (SERS) offers a relatively fast and inexpensive method for the detection of MDAI at low concentrations. Careful optimisation of the silver sol, and salt concentrations was undertaken to ensure the SERS analysis was both reproducible and sensitive. The optimised system demonstrated acceptable peak variations of less than 15% RSD and resulted in a detection limit of just 8 ppm (5.4 × 10(-5) M).

Determination of metal ion concentrations by SERS using 2,2'-bipyridyl complexes.

Surface enhanced Raman scattering (SERS) can generate characteristic spectral "fingerprints" from metal complexes, thus providing the potential for the development of methods of analysis for the identification and quantitation of a range of metal ions in solution. The advantages include sensitivity and the use of one ligand for several metals without the need for a specific chromophore. Aqueous solutions of Fe(II), Ni(II), Zn(II), Cu(II), Cr(III) and Cd(II) in the presence of excess 2,2'-bipyridyl (bipy) were analysed using SERS. Specific marker bands enabled the identification of each metal ion and the limit of detection for each metal ion was estimated. Two of the ions, Zn(II) and Cu(II), could be detected below the World Health Organisation's (WHO) recommended limits for drinking water at levels of 0.22 and 0.6 mg L(-1), respectively.

Efficient production of uniform gold nanoparticles via a streamlined low-cost, semi-automated, open-source platform.

In the quest to discover dependable and repeatable methods for producing noble metal nanospheres, both commercial and academic scientists have shown great interest. The challenge of precisely controlling the size of these nanospheres is critical, as variations can alter their optical characteristics, leading to complications in subsequent applications. In this context, we present the design and validation of an affordable, semi-automated device that synthesizes gold nanoparticles using the Turkevich method. This device, named 'NanoSynth Mini' and powered by Raspberry Pi, demonstrates the capability to generate gold nanoparticles with diameters ranging from 15 to 60 nanometers with minimal variability. Its design allows for seamless integration into lab processes, providing consistent support for extensive research initiatives.

DNA-directed formation of plasmonic core-satellite nanostructures for quantification of hepatitis C viral RNA.

Hepatitis C virus (HCV) continues to be a significant public health challenge, affecting an estimated 71 million people globally and posing risks of severe liver diseases. Despite advancements in treatments, diagnostic limitations hinder the global elimination efforts targeted by 2030. This study introduces an innovative diagnostic approach, integrating catalytic hairpin assembly (CHA) with plasmonic core-satellite gold nanoparticle (AuNP) assemblies, to enable sensitive and specific detection of HCV RNA. We optimized the stoichiometry of DNA hairpins to form highly stable three-way junctions (3WJs), minimizing non-specific reactions in an enzyme-free, isothermal amplification process. The resulting dual-transduction biosensor combines colorimetric and surface-enhanced Raman spectroscopy (SERS) techniques, utilizing the Raman reporter malachite green isothiocyanate (MGITC) for signal generation. Our system targets a conserved 23-nucleotide sequence within the HCV 5'-UTR, essential for RNA replication, facilitating pan-genotypic HCV detection that complements direct-acting antiviral strategies. We evaluated the biosensor's efficacy using fluorescence spectroscopy, native PAGE, AFM, and TEM. Findings indicate that the 60 nm core AuNPs surrounded by 20 nm satellite AuNPs achieved a ten-fold increase in sensitivity over the 10 nm satellites, detecting HCV RNA concentrations as low as 1.706 fM. This sensitivity is crucial, given the extremely low viral loads present during early infection stages. Our research demonstrates the promise of enzyme-free molecular biosensors for HCV, with the potential to provide cost-efficient, rapid, point-of-care testing, although further sensitivity enhancements are needed to address the challenges of early-stage detection.

Freeze-Driven Synthesis of DNA Hairpin-Conjugated Gold Nanoparticle Biosensors for Dual-Mode Detection.

Freeze-based immobilization of deoxyribonucleic acid (DNA) oligonucleotides on gold nanoparticles (AuNPs) is highly efficient for single-stranded oligonucleotides but typically does not accommodate structures such as snap-cooled DNA hairpins (Sc-HPs) and snap-cooled molecular beacons (Sc-MBs) frequently used for biorecognition applications. Recognizing this limitation, we have developed a modified, freeze-based technique specifically designed to enable the adsorption of such hairpin oligonucleotides onto AuNP surfaces while ensuring that they retain their biosensing capabilities. Successful hairpin oligonucleotide conjugation of varying lengths to a wide range of AuNP diameters was corroborated by dynamic light scattering, ζ-potential, and UV-vis spectrophotometry. Moreover, we conducted a thorough evaluation of this modified method, confirming the retention of the sensing functions of Sc-HPs and Sc-MBs. This advancement not only offers a more efficient route for DNA hairpin conjugation but also elucidates the underlying biorecognition functions, with implications for broader applications in molecular diagnostics.

In vivo imaging using surface enhanced spatially offset raman spectroscopy (SESORS): balancing sampling frequency to improve overall image acquisition.

In the field of optical imaging, the ability to image tumors at depth with high selectivity and specificity remains a challenge. Surface enhanced resonance Raman scattering (SERRS) nanoparticles (NPs) can be employed as image contrast agents to specifically target cells in vivo; however, this technique typically requires time-intensive point-by-point acquisition of Raman spectra. Here, we combine the use of "spatially offset Raman spectroscopy" (SORS) with that of SERRS in a technique known as "surface enhanced spatially offset resonance Raman spectroscopy" (SESORRS) to image deep-seated tumors in vivo. Additionally, by accounting for the laser spot size, we report an experimental approach for detecting both the bulk tumor, subsequent delineation of tumor margins at high speed, and the identification of a deeper secondary region of interest with fewer measurements than are typically applied. To enhance light collection efficiency, four modifications were made to a previously described custom-built SORS system. Specifically, the following parameters were increased: (i) the numerical aperture (NA) of the lens, from 0.2 to 0.34; (ii) the working distance of the probe, from 9 mm to 40 mm; (iii) the NA of the fiber, from 0.2 to 0.34; and (iv) the fiber diameter, from 100 µm to 400 µm. To calculate the sampling frequency, which refers to the number of data point spectra obtained for each image, we considered the laser spot size of the elliptical beam (6 × 4 mm). Using SERRS contrast agents, we performed in vivo SESORRS imaging on a GL261-Luc mouse model of glioblastoma at four distinct sampling frequencies: par-sampling frequency (12 data points collected), and over-frequency sampling by factors of 2 (35 data points collected), 5 (176 data points collected), and 10 (651 data points collected). In comparison to the previously reported SORS system, the modified SORS instrument showed a 300% improvement in signal-to-noise ratios (SNR). The results demonstrate the ability to acquire distinct Raman spectra from deep-seated glioblastomas in mice through the skull using a low power density (6.5 mW/mm2) and 30-times shorter integration times than a previous report (0.5 s versus 15 s). The ability to map the whole head of the mouse and determine a specific region of interest using as few as 12 spectra (6 s total acquisition time) is achieved. Subsequent use of a higher sampling frequency demonstrates it is possible to delineate the tumor margins in the region of interest with greater certainty. In addition, SESORRS images indicate the emergence of a secondary tumor region deeper within the brain in agreement with MRI and H&E staining. In comparison to traditional Raman imaging approaches, this approach enables improvements in the detection of deep-seated tumors in vivo through depths of several millimeters due to improvements in SNR, spectral resolution, and depth acquisition. This approach offers an opportunity to navigate larger areas of tissues in shorter time frames than previously reported, identify regions of interest, and then image the same area with greater resolution using a higher sampling frequency. Moreover, using a SESORRS approach, we demonstrate that it is possible to detect secondary, deeper-seated lesions through the intact skull.

Optimization of parameters for the quantitative surface-enhanced Raman scattering detection of mephedrone using a fractional factorial design and a portable Raman spectrometer.

A new optimization strategy for the SERS detection of mephedrone using a portable Raman system has been developed. A fractional factorial design was employed, and the number of statistically significant experiments (288) was greatly reduced from the actual total number of experiments (1722), which minimized the workload while maintaining the statistical integrity of the results. A number of conditions were explored in relation to mephedrone SERS signal optimization including the type of nanoparticle, pH, and aggregating agents (salts). Through exercising this design, it was possible to derive the significance of each of the individual variables, and we discovered four optimized SERS protocols for which the reproducibility of the SERS signal and the limit of detection (LOD) of mephedrone were established. Using traditional nanoparticles with a combination of salts and pHs, it was shown that the relative standard deviations of mephedrone-specific Raman peaks were as low as 0.51%, and the LOD was estimated to be around 1.6 µg/mL (9.06 × 10(-6) M), a detection limit well beyond the scope of conventional Raman and extremely low for an analytical method optimized for quick and uncomplicated in-field use.

2p or not 2p: tuppence-based SERS for the detection of illicit materials.

Deposition of silver onto British 2p coins has been demonstrated as an efficient and cost effective approach to producing substrates capable of promoting surface enhanced Raman scattering (SERS). Silver application to the copper coins is undemanding taking just 20 s, and results in the formation of multiple hierarchial dendritic structures. To demonstrate that the silver deposition sites were capable of SERS the highly fluorescent Rhodamine 6G (R6G) probe was used. Analyses indicated that Raman enhancement only occurs at the silver deposition sites and not from the roughened copper surface. The robustness of the substrate in the identification and discrimination of illegal and legal drugs of abuse was then explored. Application of the drugs to the substrates was carried out using spotting and soaking methodologies. Whilst little or no SERS spectra of the drugs were generated upon spotting, soaking of the substrate in a methanolic solution of the drugs yielded a vast amount of spectral information. Excellent reproducibility of the SERS method and classification of three of the drugs, 4-methylmethcathinone (mephedrone), 5,6-methylenedioxy-2-aminoindane (MDAI) and 3,4-methylenedioxy-N-methylamphetamine (MDMA) were demonstrated using principal components analysis and partial least squares.

Exploring the Clinical Utility of Raman Spectroscopy for Point-of-Care Cardiovascular Disease Biomarker Detection.

A variety of innovative point-of-care (POC) solutions using Raman systems have been explored. However, the vast effort is in assay development, while studies of the characteristics required for Raman spectrometers to function in POC applications are lacking. In this study, we tested and compared the performance of eight commercial Raman spectrometers ranging in size from benchtop Raman microscopes to portable and handheld Raman spectrometers using paper fluidic cartridges, including their ability to detect cardiac troponin I and heart fatty acid binding protein, both of which are well-established biomarkers for evaluating cardiovascular health. Each spectrometer was evaluated in terms of excitation wavelength, laser characteristics, and ease of use to investigate POC utility. We found that the Raman spectrometers equipped with 780 and 785 nm laser sources exhibited a reduced background signal and provided higher sensitivity compared to those with 633 and 638 nm laser sources. Furthermore, the spectrometer equipped with the single acquisition line readout functionality showed improved performance when compared to the point scan spectrometers and allowed measurements to be made faster and easier. The portable and handheld spectrometers also showed similar detection sensitivity to the gold standard instrument. Lastly, we reduced the laser power for the spectrometer with single acquisition line readout capability to explore the system performance at a laser power that change the classification from a Class 3B laser device to a Class 3R device and found that it showed comparable performance. Overall, these findings show that portable Raman spectrometers have the potential to be used in POC settings with accuracy comparable to laboratory-grade instruments, are relatively low-cost, provide fast signal readout, are easy to use, and can facilitate access for underserved communities.

The optimisation of facile substrates for surface enhanced Raman scattering through galvanic replacement of silver onto copper.

A fast and cost-effective approach for the synthesis of substrates used in surface enhanced Raman scattering (SERS) has been developed using galvanic displacement. Deposition of silver onto commercially available Cu foil has resulted in the formation of multiple hierarchical structures, whose morphology show dependence on deposition time and temperature. Analysis of the surface structure by scanning electron microscopy revealed that the more complex silver structures correlated well with increased deposition time and temperature. Using Rhodamine 6G (R6G) as a model Raman probe it was also possible to relate the substrate morphology directly with subsequent SERS intensity from the R6G analyte as well as the reproducibility across a total of 15 replicate Raman maps (20 × 20 pixels) consisting of 400 spectra at a R6G concentration of 10(-4) M. The substrate with the highest reproducibility was then used to explore the limit of detection and this compared very favourably with colloidal-based SERS assessments of the same analyte.

3D Printed SERS-Active Thin-Film Substrates Used to Quantify Levels of the Genotoxic Isothiazolinone.

Several reports present methods to fabricate thin-film substrates capable of surface-enhanced Raman scattering (SERS). Substrates synthesized by displacing silver onto copper using facile synthesis methods such as galvanic displacement can generate high levels of SERS enhancement rivaling commercially available substrates manufactured by lithographic methods. Here, we describe the optimization of a novel set of SERS-active thin-film substrates synthesized via the electroless displacement of Ag onto the surface of three-dimensional (3D) printed disks composed of the copper/polymer (PLA) composite filament. The effect of AgNO3 concentration on the deposition, morphology, and overall SERS activity of the substrates has been carefully studied. Two commonly used Raman reporters, 4-mercaptobenzoic acid (MBA) and malachite green isothiocyanate (MGITC), were used to measure the SERS output of the substrates. Good SERS signal reproducibility (RSD ∼16.8%) was measured across the surface of replicate substrates and high-sensitivity detection of MBA was achieved (10-12 M). To test the real-world application of our substrates, we opted to detect 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), which is a genotoxic, biocide common in many household products, known to leach into water supplies. Our newly developed SERS-active substrates could detect CMIT down to 10 ppm when spiked in simulated lake water samples, which is well within current agency standards.