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SignificancePoint-of-care (POC) platforms utilizing optical biosensing strategies can achieve on-site detection of biomarkers to improve the quality of care for patients in low-resource settings.AimWe aimed to develop a portable, multi-modal spectroscopic platform capable of performing Raman and fluorescence measurements from a single sample site.ApproachWe designed the spectroscopic platform in OpticStudio using commercial optical components and built the system on a portable optical breadboard. Two excitation and collection arms were utilized to detect the two optical signals. The multi-modal functionality was validated using ratiometric Raman/fluorescence samples, and the potential utility was demonstrated using a model bioassay for cardiac troponin I.ResultsThe designed spectroscopic platform achieved a spectral resolution of 0.67 ± 0.2 nm across the Raman detection range (660 to 770 nm). The ratiometric Raman/fluorescence samples demonstrated no crosstalk between the two detector arms across a gradient of high molar concentrations. Testing of the model bioassay response showed that the integrated approach improved the linearity of the calibration curve from (R2 = 0.977) for the Raman only and (R2 = 0.972) for the fluorescence only to (R2 = 0.988) for the multi-modal approach.ConclusionThese findings demonstrate the potential impact of a multi-modal POC spectroscopic platform to improve the sensitivity and robustness necessary for biomarker detection.Point-of-care (POC) platforms utilizing optical biosensing strategies can achieve on-site detection of biomarkers to improve the quality of care for patients in low-resource settings.We aimed to develop a portable, multi-modal spectroscopic platform capable of performing Raman and fluorescence measurements from a single sample site.We designed the spectroscopic platform in OpticStudio using commercial optical components and built the system on a portable optical breadboard. Two excitation and collection arms were utilized to detect the two optical signals. The multi-modal functionality was validated using ratiometric Raman/fluorescence samples, and the potential utility was demonstrated using a model bioassay for cardiac troponin I.The designed spectroscopic platform achieved a spectral resolution of 0.67 ± 0.2 nm across the Raman detection range (660 to 770 nm). The ratiometric Raman/fluorescence samples demonstrated no crosstalk between the two detector arms across a gradient of high molar concentrations. Testing of the model bioassay response showed that the integrated approach improved the linearity of the calibration curve from (RThese findings demonstrate the potential impact of a multi-modal POC spectroscopic platform to improve the sensitivity and robustness necessary for biomarker detection.Point-of-care (POC) platforms utilizing optical biosensing strategies can achieve on-site detection of biomarkers to improve the quality of care for patients in low-resource settings.We aimed to develop a portable, multi-modal spectroscopic platform capable of performing Raman and fluorescence measurements from a single sample site.We designed the spectroscopic platform in OpticStudio using commercial optical components and built the system on a portable optical breadboard. Two excitation and collection arms were utilized to detect the two optical signals. The multi-modal functionality was validated using ratiometric Raman/fluorescence samples, and the potential utility was demonstrated using a model bioassay for cardiac troponin I.The designed spectroscopic platform achieved a spectral resolution of 0.67 ± 0.2 nm across the Raman detection range (660 to 770 nm). The ratiometric Raman/fluorescence samples demonstrated no crosstalk between the two detector arms across a gradient of high molar concentrations. Testing of the model bioassay response showed that the integrated approach improved the linearity of the calibration curve from (RThese findings demonstrate the potential impact of a multi-modal POC spectroscopic platform to improve the sensitivity and robustness necessary for biomarker detection.

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.

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)., Author summary Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a disease caused by the parasite Trypanosoma brucei and has been responsible for the death of millions of people across Africa in the 20th century. It is also a major economic burden for countries endemic for trypanosomiasis, affecting livestock productivity in rural areas (Animal African Trypanosomiasis). A long-term international collaboration with the help of the World Health Organisation has resulted in the rate of human infection decreasing to less than 1000 new cases per year. However, the human disease continues to spread within remote villages. Current diagnosis is based on the detection of parasites in blood and serum samples, but this is challenging during chronic human infections with low or non-detectable parasitaemia. However, the recent discovery of extravascular skin-dwelling trypanosomes indicates that a reservoir of infection remains undetected, threatening the effort to eliminate the disease. In this study we have targeted the skin as a site for diagnosis using Raman spectroscopy and demonstrate that this method showed great promise in the laboratory, laying the foundation for field studies to examine its potential to strengthen current diagnostic strategies for detecting HAT cases.

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.

Surface-enhanced Raman scattering (SERS) is a sensitive analytical technique capable of magnifying the vibrational intensity of molecules adsorbed onto the surface of metallic nanostructures. Various solution-based SERS-active metallic nanostructures have been designed to generate substantial SERS signal enhancements. However, most of these SERS substrates rely on the chemical aggregation of metallic nanostructures to create strong signals. While this can induce high SERS intensities through plasmonic coupling, most chemically aggregated assemblies suffer from poor signal reproducibility and reduced long-term stability. To overcome these issues, here we report for the first time the synthesis of gold core–satellite nanoparticles (CSNPs) for robust SERS signal generation. The novel CSNP assemblies consist of a 30 nm spherical gold core linked to 18 nm satellite particles via linear heterobifunctional thiol–amine terminated PEG chains. We explore the effects that the varying chain lengths have on SERS hot-spot generation, signal reproducibility and long-term activity. The chain length was varied by using PEGs with different molecular weights (1000 Da, 2000 Da, and 3500 Da). The CSNPs were characterized via UV-Vis spectrophotometry, transmission electron microscopy (TEM), ζ-potential measurements, and lastly SERS measurements. The versatility of the synthesized SERS-active CSNPs was revealed through characterization of optical stability and SERS enhancement at 0, 1, 3, 5, 7 and 14 days.

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.

Human immunodeficiency virus (HIV) weakens a person’s immune system by destroying cells which are important to fighting the disease and infection. HIV is a major global public health issue with an estimated 38 million people living with HIV at the end of 2019. There is no cure for HIV. However, increased access to effective prevention, diagnosis and treatment has enabled people living with HIV to lead healthy lives. Rapid diagnosis forms an important part of the WHO’s strategy for ensuring those who are HIV positive have immediate access to care. Perhaps more critical however is that effective diagnostic testing informs individuals of their HIV status, reducing the risk of transmission. HIV prevalence within the US is disproportionately high amongst Black/African American and Hispanic/Latino American populations who often have limited access to advanced medical clinics. In this paper, we present a lateral flow device designed to detect miR-150-5p; an emerging biomarker of HIV. Based on our preliminary results shown here, we are capable of detecting the miRNA sequence at sub-ng uL-1 using colorimetric analysis, without prior amplification of the target material. We have also detailed our initial results obtained from surface-enhanced Raman scattering measurements made at the device capture line, whilst the standard deviations are large, the technique shows lots of promise for lowering the detection limit in the future.

Multiplexed assays are essential for the detection of biomarker panels. Differentiating signals from different biomarkers in a single test zone makes the detection more efficient. In this paper, a new method is designed for the synthesis of gap-enhanced nanoparticles (GeNPs) using Raman reporter molecules (RRM) and 6-amino-1-hexanethiol (6-AHT) as the spacer. The GeNPs show a nanometer-size gap, generate strong surface-enhanced Raman scattering (SERS) attributed to the gap, and exhibit discriminative spectral peaks. The strong Au-S bonds on both core and shell sides and the covalent bond between RRM and 6-AHT led to a stable structure, which ensured the stable SERS signal generation from the GeNPs. Using the GeNPs, a spectrally multiplexed assay for the detection of a biomarker panel is developed. The biomarker panel is composed of cardiac troponin I (cTnI), copeptin, and heart-type fatty acid-binding protein (h-FABP), which improves myocardial infarction (MI) diagnostic performance. A paper-based platform that is more amenable to point-of-care diagnostic analysis is used. The developed single biomarker assay achieves limits of detection of 0.01 ng mL

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.

In order to improve patient survival and reduce the amount of unnecessary and traumatic biopsies, non-invasive detection of cancerous tumours is of imperative and urgent need. Multicellular tumour spheroids (MTS) can be used as an ex vivo cancer tumour model, to model in vivo nanoparticle (NP) uptake by the enhanced permeability and retention (EPR) effect. Surface enhanced spatially offset Raman spectroscopy (SESORS) combines both surface enhanced Raman spectroscopy (SERS) and spatially offset Raman spectroscopy (SORS) to yield enhanced Raman signals at much greater sub-surface levels. By utilizing a reporter that has an electronic transition in resonance with the laser frequency, surface enhanced resonance Raman scattering (SERRS) yields even greater enhancement in Raman signal. Using a handheld SORS spectrometer with back scattering optics, we demonstrate the detection of live breast cancer 3D multicellular tumour spheroids (MTS) containing SERRS active NPs through 15 mm of porcine tissue. False color 2D heat intensity maps were used to determine tumour model location. In addition, we demonstrate the tracking of SERRS-active NPs through porcine tissue to depths of up to 25 mm. This unprecedented performance is due to the use of red-shifted chalcogenpyrylium-based Raman reporters to demonstrate the novel technique of surface enhanced spatially offset resonance Raman spectroscopy (SESORRS) for the first time. Our results demonstrate a significant step forward in the ability to detect vibrational fingerprints from a tumour model at depth through tissue. Such an approach offers significant promise for the translation of NPs into clinical applications for non-invasive disease diagnostics based on this new chemical principle of measurement.

Spatially offset Raman spectroscopy (SORS) provides chemical analysis at depth even when obscuring barriers such as plastic or tissue are present. As the collection probe is moved further away from the point of laser excitation, scattered photons from deeper layers begin to dominate the acquired spectra, thus giving rise to through barrier detection. Here, we demonstrate the potential of conventional Raman (CR) and SORS for through barrier detection using handheld spectrometers. We report the collection of Raman signals from an ethanol solution through plastic at thicknesses of up to 21 mm using SORS in combination with multivariate analysis. SORS is compared to CR, where we also demonstrate impressive through barrier detection of ethanol at depths up to 9 mm. We also highlight the advantage of applying multivariate analysis for through barrier detection using CR or SORS, particularly when peaks with similar spectral features are present in both the barrier and analyte spectra. In addition, to the best of our knowledge, this is the first report of the assessment of the maximum level of through barrier detection using handheld CR and SORS instruments with a backscattering geometry.

The photothermal release of single stranded DNA (ssDNA) from the surface of gold nanoparticles of different shapes and sizes is a promising mode of delivering DNA for gene-therapy applications. Here, we demonstrate the first targeted photothermal release of ssDNA from hollow gold nanospheres (HGNs) and analyse the release of the ssDNA using quantitative surface enhanced Raman scattering (SERS). The HGNs used demonstrate a tunable localized surface plasmon resonance (LSPR) frequency while maintaining size consistency, allowing for selective ssDNA release based on matching the excitation frequency to the plasmon resonance. It is shown that HGNs with resonances at 760 and HGN 670 nm release significant amounts of ssDNA when excited via 785 nm and 640 nm lasers respectively. When excited with a wavelength far from the LSPR of the particles, the ssDNA release is negligible. This is the first demonstration of SERS to analyze the amount of ssDNA photothermally released from the surface of HGNs. In contrast to traditional fluorescence measurements, this SERS based approach provides quantitatively robust data for analysis of ssDNA release and lays a strong foundation for future studies exploiting plasmonically induced ssDNA release.

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.

Through utilizing the depth penetration capabilities of SESORS, multiplexed imaging and classification of three singleplex nanotags and a triplex of nanotags within breast cancer tumour models is reported for the first time through depths of 10 mm using a handheld SORS instrument.

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.

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).

Conjugation of aptamers and their corresponding analytes onto plasmonic nanoparticles mediates the formation of nanoparticle assemblies: molecularly bound nanoclusters which cause a measurable change in the colloid’s optical properties. In this paper the optimization of a surface enhanced Raman spectroscopy (SERS) competitive binding assay is presented utilizing plasmonic ‘target’ and magnetic ‘probe’ nanoparticles for the detection of the toxin bisphenol-A (BPA). These assay nanoclusters were housed inside three types of optofluidic chips patterned with magnetically-activated nickel pads, in either a straight or array pattern. Both Fe2O3 and Fe2CoO4 were compared as potential magnetic cores for the silver coated probe nanoparticles. We found that the Ag@ Fe2O3 particles were, on average, more uniform in size and more stable than Ag@ Fe2CoO4, whereas the addition of cobalt significantly improved the collection time of particles. Using Raman mapping of the assay housed within the magneto-fluidic chips, it was determined that a 1 x 5 array of 50 µm square nickel pads provided the most uniform SERS enhancement of the assay (coefficient of variation ~25%) within the magneto-fluidic chip. Additionally, the packaged assay demonstrated the desired response to BPA, verifying the technology’s potential to translate magnetic nanoparticle assays into a user-free optical analysis platform.

We report a novel approach for patterning thermoresponsive hydrogels based on N,N-diethylacrylamide (DEAAm) and bifunctional Jeffamine ED-600 by dip-pen nanolithography (DPN). The direct writing of micron-sized thermoresponsive polymer spots was achieved with efficient control over feature size. A Jeffamine-based ink prepared through the combination of organic polymers, such as DEAAm, in an inorganic silica network was used to print thermosensitive arrays on a thiol-silanised silicon oxide substrate. The use of a Jeffamine hydrogel, acting as a carrier matrix, allowed a reduction in the evaporation of ink molecules with high volatility, such as DEAAm, and facilitated the transfer of ink from tip to substrate. The thermoresponsive behaviour of polymer arrays which swell/de-swell in aqueous solution in response to a change in temperature was successfully characterised by atomic force microscopy (AFM) and Raman spectroscopy: a thermally-induced change in height and hydration state was observed, respectively. Finally, we demonstrate that cells can adhere to and interact with these dynamic features and exhibit a change in behaviour when cultured on the substrates above and below the transition temperature of the Jeffamine/DEAAm thermoresponsive hydrogels. This demonstrates the potential of these micropatterned hydrogels to act as a controllable surface for cell growth.

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 (C16H16N2O2) 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

Conjugation of oligonucleotides or aptamers and their corresponding analytes onto plasmonic nanoparticles mediates the formation of nanoparticle assemblies: molecularly bound bundles of nanoparticles which cause a measurable change in the colloid's optical properties. Here, we present further optimization of a "SERS off" competitive binding assay utilizing plasmonic and magnetic nanoparticles for the detection of the toxin bisphenol A (BPA). The assay involves 1) a 'target' silver nanoparticle functionalized with a Raman reporter dye and PEGylated BPA-binding DNA aptamers, and 2) a version of the toxin BPA, bisphenol A diglycidyl ether (BADGE), PEGylated and immobilized onto a silver coated magnetic 'probe' nanoparticle. When mixed, these target and probe nanoparticles cluster into magnetic dimers and trimers and an enhancement in their SERS spectra is observed. Upon introduction of free BPA in its native form, target AgNPs are competitively freed; reversing the nanoparticle assembly and causing the SERS signal to "turn-off" and decrease in response to the competitive binding event. The assay particles were housed inside two types of optofluidic chips containing magnetically active nickel pads, in either a straight or spotted pattern, and both Fe2O3 and Fe2CoO4 were compared as magnetic cores for the silver coated probe nanoparticle. We found that the Ag@ Fe2O3 particles were, on average, more uniform in size and more stable than Ag@ Fe2CoO4, while the addition of cobalt significantly improved the collection time of particles within the magnetic chips. Using 3D Raman mapping, we found that the straight channel design with the Ag@ Fe2O3 particles provided the most uniform nanoparticle organization, while the spotted channel design with Ag@ Fe2CoO4 demonstrated a larger SERS enhancement, and thus a lower limit of detection.

We report a novel nanophotonic biosensor surface capable of both colorimetric detection and Raman-scattered detection of DNA infection markers at extreme sensitivities. Combining direct-write lithography, dip-pen nanolithography based DNA patterning, and molecular self-assembly, we create molecularly-active plasmonic nanostructures onto which metallic nanoparticles are located via DNA-hybridization. Arraying these structures enables optical surfaces that change state when contacted by specific DNA sequences; shifting the surface color while simultaneously generating strong Raman-scattering signals. Patterning the DNA markers onto the plasmonic surface as micro-scale symbols results in easily identifiable color shifts, making this technique applicable to multiplexed lab-on-a-chip and point-of-care diagnostic applications.

The synergy afforded by the combination of biofunctionalised nanoparticles and surface enhanced Raman scattering (SERS) has expanded the analytical toolbox for clinical diagnostics and therapeutics. Since their inception, SERS-active nanoparticles have been developed into biofunctional nanoparticles (BFNPs) using a variety of methods to attach biomolecules and pacification layers to nanoparticles to enable detection of various diseases or cancers in vitro and in vivo. However, while there are many reports of the use of BFNPs for diagnostic or therapeutic applications, very few are implemented in a “real” clinical setting, for example, detection of disease biomarkers in tissue or the delivery of drugs to affected cells. This review covers recent advances made in the development of BFNPs for SERS-based detection of clinical samples using in vitro and in vivo methods.

Two advances in the development of a one-pot method to prepare silver nanoparticles (AgNPs) using the Tollens' reagent are described. First, a template-directed process of AgNP synthesis using resorcinol triazole ligands bearing two pendent galactose sugars is shown. Second, the conversion of these AgNPs into SERS nanotags is demonstrated using malachite green isothiocyanate as the Raman reporter molecule.

Functionalized colloidal nanoparticles for SERS serve as a promising multifunctional assay component for blood biomarker detection. Proper design of these nanoprobes through conjugation to spectral tags, protective polymers, and sensing ligands can provide experimental control over the sensitivity, range, reproducibility, particle stability, and integration with biorecognition assays. Additionally, the optical properties and degree of electromagnetic SERS signal enhancement can be altered and monitored through tuning the nanoparticle shape, size, material and the colloid's local surface plasmon resonance (LSPR). Aptamers, synthetic affinity ligands derived from nucleic acids, provide a number of advantages for biorecognition of small molecules and toxins with low immunogenicity. DNA aptamers are simpler and more economical to produce at large scale, are capable of greater specificity and affinity than antibodies, are easily tailored to specific functional groups, can be used to tune inter-particle distance and shift the LSPR, and their intrinsic negative charge can be utilized for additional particle stability.1,2 Herein, a "turn-off" competitive binding assay platform involving two different plasmonic nanoparticles for the detection of the toxin bisphenol A (BPA) using SERS is presented. A derivative of the toxin is immobilized onto a silver coated magnetic nanoparticle (Ag@MNP), and a second solid silver nanoparticle (AgNP) is functionalized with the BPA aptamer and a Raman reporter molecule (RRM). The capture (Ag@MNP) and probe (AgNP) particles are mixed and the aptamer binding interaction draws the nanoparticles closer together, forming an assembly that results in an increased SERS signal intensity. This aptamer mediated assembly of the two nanoparticles results in a 100x enhancement of the SERS signal intensity from the RRM. These pre-bound aptamer/nanoparticle conjugates were then exposed to BPA in free solution and the competitive binding event was monitored by the decrease in SERS intensity.

Bacterial meningitis is well known for its rapid onset and high mortality rates, therefore rapid detection of bacteria found in cerebral spinal fluid (CSF) and subsequent effective treatment is crucial. A new quantitative assay for detection of three pathogens that result in bacterial meningitis using a combination of lambda exonuclease (λ-exonuclease) and surface enhanced Raman scattering (SERS) is reported. SERS challenges current fluorescent-based detection methods in terms of both sensitivity and more importantly the detection of multiple components in a mixture, which is becoming increasingly more desirable for clinical diagnostics. λ-Exonuclease is a processive enzyme that digests one strand of double stranded DNA bearing a terminal 5′-phosphate group. The new assay format involves the simultaneous hybridisation of two complementary DNA probes (one containing a SERS active dye) to a target sequence followed by λ-exonuclease digestion of double stranded DNA and SERS detection of the digestion product. Three meningitis pathogens were successfully quantified in a multiplexed test with calculated limits of detection in the pico-molar range, eliminating the need for time consuming culture based methods that are currently used for analysis. Quantification of each individual pathogen in a mixture using SERS is complex, however, this is the first report that this is possible using the unique spectral features of the SERS signals combined with partial least squares (PLS) regression. This is a powerful demonstration of the ability of this SERS assay to be used for analysis of clinically relevant targets with significant advantages over existing approaches and offers the opportunity for future deployment in healthcare applications.

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.

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.