Preeclampsia (PE) is a condition that affects women during pregnancy. It is a leading cause of maternal and infant death worldwide and is typically detected in the second trimester or later. Predicting PE at an early stage is one of the most important goals of maternal-fetal medicine. Thus, there is an unmet need for accurate early detection of preeclampsia, ideally at the point-of-care. In a pilot study, microRNA-20a (miR-20a) showed an upregulation in the blood samples of the first trimester of preeclamptic pregnancies when compared to healthy mothers. In this research, a dual capture probe sandwich assay, sensitive to miR-20a, that used surface-enhanced Raman scattering (SERS)-active gold nanoparticles for the signal transduction was developed. The assay was translated to a lateral flow paper fluidic device, for potential low cost and ease of use in low-resource settings. Each paper layer has different pore sizes and features an array of buffers that store nanoparticle conjugates, guide flow, and reduce non-specific binding. Using a handheld Raman reader with a 638 nm excitation laser, reliable SERS detection of miR-20a was shown with resolutions down to 1 nM. Colorimetric detection of these concentrations using the RGB pixels from scanning the test line is also depicted, showing multi-modal detection to potentially enhance specificity for this early epigenetic biomarker.
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.
Today, approximately 5 million Americans are living with Congestive Heart Failure (CHF), and this burden is higher for African American communities, even for control groups. Implantable devices to measure pulmonary artery pressure are sometimes indicated as a surrogate measure for fluid retention in the lungs caused by worsening heart failure but are often inaccessible to underserved communities due to the prohibitive costs of the device and surgical procedure. Thus, we are implementing a sensitive immunoassay on a low-cost paper fluidic platform, to enable the frequent and easy measurements of B-type Natriuretic Peptide (BNP), an important biomarker secreted by cardiomyocytes in response to the increasing ventricular stretch and cardiac volume. BNP concentrations are often used to classify CHF into 4 levels of severity on the New York Heart Association (NYHA) scale. The paper fluidic cartridge is composed of thin, economical cotton, glass fiber and nitrocellulose membranes, that are modified to improve the flow of the sample volume. We incorporate a sandwich assay using surface-enhanced Raman scattering (SERS)-active gold nanoparticles for signal transduction, a BNP-specific aptamer for detection as well as stabilization of the nanoparticle conjugate, and a monoclonal antibody specific to BNP for the recognition element, onto this cartridge. By using a handheld Raman reader with a 638 nm excitation laser, we retrieve SERRS spectra for the malachite green isothiocyanate dye which corresponds to BNP capture ranging from 0.3 ng/mL (Stage II) to 1 ng/mL (Stage IV). We also show the visual, colorimetric detection of these concentrations using the RGB pixels from the test line, demonstrating the potential for a multi-modal approach for this diagnostic test.
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.
Conjugation of aptamers and their corresponding analytes onto plasmonic nanoparticles mediates the formation of nanoparticle assemblies: molecularly bound nanoclusters that cause a measurable change in the colloid’s optical properties. The optimization of a surface-enhanced Raman spectroscopy (SERS) competitive binding assay utilizing plasmonic “target” and magnetic “probe” nanoparticles for the detection of the toxin bisphenol-A (BPA) is presented. 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 magnetofluidic chips, it was determined that a 1×5 array of 50 μm square nickel pads provided the most uniform SERS enhancement of the assay (coefficient of variation ∼25%) within the magnetofluidic 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 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.
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.
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.
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