Surface-Enhanced Raman spectroscopy for Point-of-Care Bioanalysis: From lab to field

Intravenous iodinated contrast is a commonly used diagnostic aid to improve image quality on computed tomography. There exists a small risk of post-contrast acute kidney injury in patients receiving IV contrast. One of the biggest risk factors for developing PC-AKI is the presence of pre-existing renal dysfunction, making it important to measure the renal function prior to contrast administration. Point of care (POC) devices offer a quick estimation of renal function, potentially improving workflows in radiology departments. Two POC devices were evaluated, the Nova StatSensor and Abbott iSTAT. Patients undergoing routine radiological investigations had blood collected and analysed by a POC method and the laboratory method (Beckman AU5800). The two values were analysed and compared. Renal function was calculated using eGFR via the CKD-EPI result. eGFR values were stratified as high risk (eGFR < 30), moderate risk (eGFR 30-59) and low risk (eGFR ≥ 60). One hundred eighty-six patients were included in the study. One hundred one patients underwent the Abbott iSTAT analysis, 139 patients underwent Nova StatSensor analysis, and 53 had both. Statistical analysis revealed that the StatSensor R2 value was 0.77, and coefficient variation was 10.65%. iSTAT had a R2 value of 0.83 and coefficient variation of 7.36%. The POC devices did not miss any high-risk patients but underreported eGFR values in certain patients. POC devices are moderately accurate at detecting renal impairment in patients undergoing radiological investigations. They seem to be a good screening tool; however, any low eGFR values should be further examined.

The urgent need for sensitive, rapid, and reliable diagnostic methodologies to control and prevent life-threatening pandemic infectious disease, such as COVID-19, remains a critical priority. Timely and on-site detection of viral pathogens is essential for effective disease management and mitigation of societal disruptions. Recent advancements in optical diagnostic methods have positioned them at the forefront of healthcare diagnostics, offering high sensitivity and specificity as viable alternatives to conventional techniques such as the Polymerase Chain Reaction (PCR), which often suffer from time delays and limited accessibility in resource-constrained environments. This review elucidates the potential of various optical diagnostic techniques, highlighting their advantages over traditional methods. It encompasses a range of optical modalities, including fluorescence-based approaches, Raman spectroscopy (RS), Plasmonic techniques (e.g., surface plasmon resonance (SPR), localized SPR, (LSPR), surface-enhanced Raman spectroscopy (SERS), and surface-enhanced fluorescence (SEF)), super resolution microscopies (SRMs), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and integrated platforms such as waveguides and molecularly imprinted polymer (MIP)-based biosensors. Additionally, the evolution of novel biosensors, particularly 5th and 6th generation biosensors, in healthcare and the challenges related to these technologies were discussed. This studies reviewed aims to advance the development of portable, sensitive, specific, and cost-effective point-of-care (POC) diagnostic devices for the rapid detection of viral pathogens.

Civil wars produce immense humanitarian crises, causing millions of individuals to seek refuge in other countries. The rate of disease prevalence has inclined among the refugees, increasing the cost of healthcare. Complex medical conditions and high numbers of patients at healthcare centers overwhelm the healthcare system and delay diagnosis and treatment. Point-of-care (PoC) testing can provide efficient solutions to high equipment cost, late diagnosis, and low accessibility of healthcare services. However, the development of PoC devices in developing countries is challenged by several barriers. Such PoC devices may not be adopted due to prejudices about new technologies and the need for special training to use some of these devices. Here, we investigated the concerns of end users regarding PoC devices by surveying healthcare workers and doctors. The tendency to adopt PoC device changes is based on demographic factors such as work sector, education, and technology experience. The most apparent concern about PoC devices was issues regarding low accuracy, according to the surveyed clinicians.

Life Sciences Discovery and Technology Highlights

SERS-based methods for the detection of genomic biomarkers of cancer

Nonspecificity and low reproducibility are always the main challenges in surface-enhanced Raman scattering (SERS) detection, especially for testing real samples. In this study, we developed a sandwich-like sensor (AuA-pMIP) to detect rhodamine 6G (R6G) by integrating a porous molecularly imprinted polymer (pMIP) with a well-ordered AuNP array (AuA). To form a uniformly distributed hot spot, AuA was fabricated at an oil-water interface and was subsequently fixed between pMIP and a support slide. Finite-difference time-domain simulation indicated that the enhanced electric field covered a distance of ∼2 μm above the AuA, in which the pMIP provided effective mass-transfer channels and sufficient specific binding sites for target molecules. High specificity for AuA-pMIP in R6G detection was demonstrated by comparing the SERS performance of R6G on AuA-pMIP with that of its structural analogues on the same sensor. Remarkably, the stable sandwich-like structure allowed us to achieve a recyclable SERS sensor with high reproducibility. Finally, AuA-pMIP displayed excellent specificity and sensitivity toward R6G in a test based on a real orange juice sample. This study presents a promising method to achieve real sample testing on a SERS platform.

ObjectivesTo assess the effect of a point of care (POC) device for testing lipids and HbA1c in addition to testing by community laboratory facilities (usual practice) on the completion of cardiovascular disease (CVD) risk assessments in general practice.MethodsWe conducted a pragmatic, cluster randomised controlled trial in 20 New Zealand general practices stratified by size and rurality and randomised to POC device plus usual practice or usual practice alone (controls). Patients aged 35–79 years were eligible if they met national guideline criteria for CVD risk assessment. Data on CVD risk assessments were aggregated using a web-based decision support programme common to each practice. Data entered into the on-line CVD risk assessment form could be saved pending blood test results. The primary outcome was the proportion of completed CVD risk assessments. Qualitative data on practice processes for CVD risk assessment and feasibility of POC testing were collected at the end of the study by interviews and questionnaire. The POC testing was supported by a comprehensive quality assurance programme.ResultsA CVD risk assessment entry was recorded for 7421 patients in 10 POC practices and 6217 patients in 10 control practices; 99.5% of CVD risk assessments had complete data in both groups (adjusted odds ratio 1.02 [95%CI 0.61–1.69]). There were major external influences that affected the trial: including a national performance target for CVD risk assessment and changes to CVD guidelines. All practices had invested in systems and dedicated staff time to identify and follow up patients to completion. However, the POC device was viewed by most as an additional tool rather than as an opportunity to review practice work flow and leverage the immediate test results for patient education and CVD risk management discussions. Shortly after commencement, the trial was halted due to a change in the HbA1c test assay performance. The trial restarted after the manufacturing issue was rectified but this affected the end use of the device.ConclusionsPerformance incentives and external influences were more powerful modifiers of practice behaviours than the POC device in relation to CVD risk assessment completion. The promise of combining risk assessment, communication and management within one consultation was not realised. With shifts in policy focus, the utility of POC devices for patient engagement in CVD preventive care may be demonstrated if fully integrated into the clinical setting.Trial registrationAustralian New Zealand Clinical Trials Registry ACTRN12613000607774

Plasmonic sensors for point of care diagnostics.

It is demonstrated that Raman Spectroscopy is a fast and sensitive tool for the detection and classification of molecular species. The vibrational spectrum inherently serves as fingerprint of the chemical composition of each sample a thus makes identification and early warning of threats possible. Also microorganisms in areas susceptible to bacterial contamination can be sensed. However, to increase the sensitivity and selectivity of the technique various solutions have been studied such as Resonant Raman Spectroscopy or Surface-Enhanced Raman Spectroscopy (SERS). In this work we present our results on the application of the SERS technique for the characterization of Bacillus atrophaeus spores, a biological and genetic simulant of the deadly bacterium Bacillus anthracis, already used in terroristic attacks in 2001 against U.S. media and government offices causing the death of five people and the infection of other 22. This work is part of the RAMBO project (Rapid Air particle Monitoring against Biological threats) whose ultimate goal is the development of an advanced sensor with high performances, capable of detecting few spores or bacilli of dangerous species with good selectivity and reliability so as to be used as an early warning sensor. In this context, the SERS technique allows to recognize the characteristics vibrational bands of the spores with a scan duration of only few seconds, on amounts of few tenths of spores or less, demonstrating how it can be considered an effective and fast technique for early warning of biological threats as it has been considered in the RAMBO project.

Complex and tedious sample preparation processes have greatly limited rapid analyses of biological samples. In this work, an all-in-one sample preparation strategy based on a miniaturized gas membrane separation/oven ring enrichment (GMS/ORE) device was developed for efficient surface enhanced Raman scattering (SERS) analyses of trace biomarkers in biofluid samples. This strategy integrating gasification separation, liquid trapping, derivatization SERS activation, and coffee-ring enrichment could highly promote the efficiency of sample preparation. Meanwhile, the edges of membranes modified by the hydrophobic-infusing slippery liquid-induced uniform "coffee-ring" effect could significantly improve the sensitivity and stability for SERS quantification. By adapting proper derivatization approaches to the miniaturized GMS/ORE pretreatment, the matrix effects in samples could be prominently eliminated, and clear SERS responses could be obtained for the selective analyses of target biomarkers. The miniaturized GMS/ORE device was practically applied for SERS analyses of trace biomarkers in biofluids, including hydrogen sulfide in saliva samples, creatinine in serum samples, and sarcosine, creatinine, and dimethyl disulfide in urine samples. Accurate quantification of all biomarkers was achieved with recoveries of 89.5%-120.0%, and the contents found by GMS/ORE-SERS matched well with those found by corresponding chromatographic methods with relative errors from -8.6% to 9.3%. The miniaturized GMS/ORE device with multiple parallel processing units could simultaneously treat eight samples in one run with a total analysis time of 40 min. Such an efficient all-in-one strategy integrated on a miniaturized device possesses great potential for fast on-site/point-of-care detection in analytical science and clinical medicine.

Considerable research effort has been directed towards developing analytical techniques for disease diagnosis, prognosis, and monitoring treatment response. These techniques involve tracking various biomarkers found in the biological fluids, such as circulating tumour cells (CTCs), small extracellular vesicles (sEVs), circulating nucleic acids (RNA and DNA), and specific proteins. Collectively, these analyses are known as liquid biopsies. However, due to the low abundance of these biomarkers in blood samples, there is an urgent need to establish rapid and reliable assays to detect their presence and quantity in patient samples. Surface-enhanced Raman scattering (SERS) has attracted significant attention for this purpose due to its unique advantages, including high sensitivity, specificity, multiplexing capability, and photostability. In SERS-related applications, plasmonic nanostructures play a pivotal role, especially in label-free sensing platforms, detecting molecular interactions at extremely low analyte concentrations. They can be functionalized with specific receptors for selective binding, allowing for sensitive and real-time detection of biomolecules like proteins or nucleic acids. Anisotropic nanostructures serve as excellent substrates for SERS, enhancing Raman signals through intense electromagnetic hotspots and enabling multiplexed assays for simultaneous detection of multiple analytes. Overall, anisotropic plasmonic nanostructures offer versatile and promising platforms for sensitive and selective biosensing applications, holding significant potential for advancements in diagnostics and biomedical research.This thesis explores properties of anisotropic nanostructures, particularly bimetallic nanostars, that can help designing improved SERS sensing platforms. The first part of the thesis details the development of a straightforward synthesis method to prepare anisotropic nanostructures and their modifications with silver layer for SERS signal enhancement. Further, this thesis addresses the limited research on the adsorption properties of aromatic thiols onto gold nanoparticles of different morphologies. It investigates the adsorption kinetics of thiolated aromatic molecules, on gold nanoparticles with various shapes using SERS. To address these differences, a universal method was proposed in the thesis to evaluate the quantity of tightly bound adsorbed molecules on GNPs regardless of particle size, shape, or concentration. This method offers a standardized approach to assess the adsorption capabilities of GNPs with different morphologies, thereby enhancing our understanding of their potential applications in various fields, particularly in analytical methods like SERS.In the later parts of the thesis the interactions between bioanalytes, such as protein and anisotropic nanoparticles, were investigated. Our work aimed to bridge the gap in understanding protein binding to non-spherical nanoparticles by investigating the differences in biomolecular interaction between spherical and anisotropic GNPs, with a specific focus on nanostars. The investigation employed a combination of experimental spectroscopic and scattering techniques (UV-Visible, SERS, small-angle X-ray scattering [SAXS], small-angle neutron scattering [SANS]) and computational techniques (finite-element method [FEM] electromagnetic wave interaction simulations and molecular dynamic [MD] simulations). Our results demonstrated drastic differences in binding patterns of protein, with nanostars demonstrating unique particle-protein structures, where protein binds to core (stabilizing the structure), leaving the plasmonic-antennas (tips) unoccupied.In the last part of the thesis, we have utilized our knowledge of interaction between nanostructures and bio-analytes to build the most efficient sensing platforms for biomarkers detection. We propose different assays for the SERS detection of bioanalytes. In particular we explore SERS-based sensing platform for the identification of bacteria strains via label-free SERS. Additionally, with label-free SERS we study protein features to distinguish recombinant and commercial human serum albumin. The work also features cellular uptake of SERS tags with nanostars as plasmonic core. Our results suggest, that nanostars based SERS tags (AuNS@MBA) caused a significant toxicity in macrophages (J774) but not the cancer cell line (MCF7), which related to the level of uptake. This finding emphasises the need for future studies that concentrate on different cell lines to develop a thorough knowledge of the outcomes.Overall, this thesis presents significant contributions to the development of nanostructures for biosensing applications and lays the groundwork for further advancements in this field.

Salmonella bacteria is a foodborne pathogen found mainly in food products causing severe symptoms in the individual, such as diarrhea, fever, and abdominal cramps after consuming the infected food, which can be fatal in some severe cases. Rapid and selective methods to detect Salmonella bacteria can prevent outbreaks when ingesting contaminated food. Nanobiosensors are a highly sensitive, simple, faster, and lower cost method for the rapid detection of Salmonella, an alternative to conventional enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) techniques. This study systematically searched and analyzed literature data related to nucleic acid-based nanobiosensors (NABs) with nanomaterials to detect Salmonella in food, retrieved from three databases, published between 2010 and 2021. We extracted data and critically analyzed the effect of nanomaterial functionalized with aptamer or DNA at the limit of detection (LOD). Among the nanomaterials, gold nanoparticles (AuNPs) were the most used nanomaterial in studies due to their unique optical properties of the metal, followed by magnetic nanoparticles (MNPs) of Fe3O4, copper nanoparticles (CuNPs), and also hybrid nanomaterials multiwalled carbon nanotubes (c-MWCNT/AuNP), QD/UCNP-MB (quantum dotes upconverting nanoparticle of magnetic beads), and cadmium telluride quantum dots (CdTe QDs@MNPs) showed excellent LOD values. The transducers used for detection also varied from electrochemical, fluorescent, surface-enhanced Raman spectroscopy (SERS), RAMAN spectroscopy, and mainly colorimetric due to the possibility of visualizing the detection result with the naked eye. Furthermore, we show the magnetic separation system capable of detecting the target amplification of the genetic material. Finally, we present perspectives, future research, and opportunities to use point-of-care (POC) diagnostic devices as a faster and lower cost approach for detecting Salmonella in food as they prove to be viable for resource-constrained environments such as field-based or economically limited conditions.

We will present surface enhanced Raman spectroscopy (SERS) and localized surface plasmon resonance (LSPR) detection of hydrogen peroxide (H2O2) using plasmonic gels. It has been known that reactive oxygen species (ROS), such as hydrogen peroxide, are involved in various biological processes, including metabolism, cell signaling, protein folding, biosynthesis, and host defense. Therefore, developing a simple and sensitive method for monitoring ROS levels is very important for live cell studies. Nevertheless, a challenge of utilizing SERS-based or LSPR-based method for molecular detection in complex fluids, such as cell culture media, is that a variety of molecules in sample solutions could interfere with detection results. In addition, when using SERS-based methods, the chance of having the target molecules at the SERS hot spots is reduced when other molecules are present in the solution. To enable detection of H2O2 in cell culture media, we have developed SERS and LSPR detection methods based on gels containing plasmonic nanoparticles. Since gels are filter-like materials, H2O2 can penetrate through the gels, but cells and large molecules such as proteins are blocked. We have successfully utilized these two methods to detect H2O2 in cell culture media without any sample pretreatment.

Food safety issues have recently attracted public concern. The deleterious effects of compromised food safety on health have rendered food safety analysis an approach of paramount importance. While conventional techniques such as high-performance liquid chromatography and mass spectrometry have traditionally been utilized for the detection of food contaminants, they are relatively expensive, time-consuming and labor intensive, impeding their use for point-of-care (POC) applications. In addition, accessibility of these tests is limited in developing countries where food-related illnesses are prevalent. There is, therefore, an urgent need to develop simple and robust diagnostic POC devices. POC devices, including paper- and chip-based devices, are typically rapid, cost-effective and user-friendly, offering a tremendous potential for rapid food safety analysis at POC settings. Herein, we discuss the most recent advances in the development of emerging POC devices for food safety analysis. We first provide an overview of common food safety issues and the existing techniques for detecting food contaminants such as foodborne pathogens, chemicals, allergens, and toxins. The importance of rapid food safety analysis along with the beneficial use of miniaturized POC devices are subsequently reviewed. Finally, the existing challenges and future perspectives of developing the miniaturized POC devices for food safety monitoring are briefly discussed.

Point of care testing: The impact of nanotechnology