Haley Marks

Rational Design of a Bisphenol A Aptamer Selective Surface- Enhanced Raman Scattering Nanoprobe

Surface-enhanced Raman scattering (SERS) optical nanoprobes offer a number of advantages for ultrasensitive analyte detection. These functionalized colloidal nanoparticles are a multifunctional assay component. providing a platform for conjugation to spectral tags, stabilizing polymers, and biorecognition elements such as aptamers or antibodies. We demonstrate the design and characterization of a SERS-active nanoprobe and investigate the nanoparticles’ biorecognition capabilities for use in a competitive binding assay. Specifically, the nanoprobe is designed for the quantification of bisphenol A (BPA) levels in the blood after human exposure to the toxin in food and beverage plastic packaging. The nanoprobes demonstrated specific affinity to a BPA aptamer with a dissociation constant Kd of 54 nM, and provided a dose-dependent SERS spectra with a limit of detection of 3 nM. Our conjugation approach shows the versatility of colloidal nanoparticles in assay development, acting as detectable spectral tagging elements and biologically active ligands concurrently.

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

Abstract Point-of-care (POC) device development is a growing field that aims to develop low-cost, rapid, sensitive in-vitro diagnostic testing platforms that are portable, self-contained, and can be used anywhere – from modern clinics to remote and low resource areas. In this review, surface enhanced Raman spectroscopy (SERS) is discussed as a solution to facilitating the translation of bioanalytical sensing to the POC. The potential for SERS to meet the widely accepted “ASSURED” (Affordable, Sensitive, Specific, User-friendly, Rapid, Equipment-free, and Deliverable) criterion provided by the World Health Organization is discussed based on recent advances in SERS in vitro assay development. As SERS provides attractive characteristics for multiplexed sensing at low concentration limits with a high degree of specificity, it holds great promise for enhancing current efforts in rapid diagnostic testing. In outlining the progression of SERS techniques over the past years combined with recent developments in smart nanomaterials, high-throughput microfluidics, and low-cost paper diagnostics, an extensive number of new possibilities show potential for translating SERS biosensors to the POC.

Rapid isolation of blood plasma using a cascaded inertial microfluidic device

Blood, saliva, mucus, sweat, sputum, and other biological fluids are often hindered in their ability to be used in point-of-care (POC) diagnostics because their assays require some form of off-site sample pre-preparation to effectively separate biomarkers from larger components such as cells. The rapid isolation, identification, and quantification of proteins and other small molecules circulating in the blood plasma from larger interfering molecules are therefore particularly important factors for optical blood diagnostic tests, in particular, when using optical approaches that incur spectroscopic interference from hemoglobin-rich red blood cells (RBCs). In this work, a sequential spiral polydimethylsiloxane (PDMS) microfluidic device for rapid (∼1 min) on-chip blood cell separation is presented. The chip utilizes Dean-force induced migration via two 5-loop Archimedean spirals in series. The chip was characterized in its ability to filter solutions containing fluorescent beads and silver nanoparticles and further using blood solutions doped with a fluorescent protein. Through these experiments, both cellular and small molecule behaviors in the chip were assessed. The results exhibit an average RBC separation efficiency of ∼99% at a rate of 5.2 × 106 cells per second while retaining 95% of plasma components. This chip is uniquely suited for integration within a larger point-of-care diagnostic system for the testing of blood plasma, and the use of multiple filtering spirals allows for the tuning of filtering steps, making this device and the underlying technique applicable for a wide range of separation applications.

Development of a miRNA surface-enhanced Raman scattering assay using benchtop and handheld Raman systems

Abstract. DNA-functionalized nanoparticles, when paired with surface-enhanced Raman spectroscopy (SERS), can rapidly detect microRNA. However, widespread use of this approach is hindered by drawbacks associated with large and expensive benchtop Raman microscopes. MicroRNA-17 (miRNA-17) has emerged as a potential epigenetic indicator of preeclampsia, a condition that occurs during pregnancy. Biomarker detection using an SERS point-of-care device could enable prompt diagnosis and prevention as early as the first trimester. Recently, strides have been made in developing portable Raman systems for field applications. An SERS assay for miRNA-17 was assessed and translated from traditional benchtop Raman microscopes to a handheld system. Three different photoactive molecules were compared as potential Raman reporter molecules: a chromophore, malachite green isothiocyanate (MGITC), a fluorophore, tetramethylrhodamine isothiocyanate, and a polarizable small molecule 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). For the benchtop Raman microscope, the DTNB-labeled assay yielded the greatest sensitivity under 532-nm laser excitation, but the MGITC-labeled assay prevailed at 785 nm. Conversely, DTNB was preferable for the miniaturized 785-nm Raman system. This comparison showed significant SERS enhancement variation in response to 1-nM miRNA-17, implying that the sensitivity of the assay may be more heavily dependent on the excitation wavelength, instrumentation, and Raman reporter chosen than on the plasmonic coupling from DNA/miRNA-mediated nanoparticle assemblies.

Comparison of Fe2O3 and Fe2CoO4 core-shell plasmonic nanoparticles for aptamer mediated SERS assays

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.

SERS-based hydrogel sensors for pH and enzymatic substrates

A sensing hydrogel for potential implantation was demonstrated using pH-sensitive surface-enhanced Raman scattering (SERS) reporters. The gold nanoparticle-based pH probes were encapsulated within polyelectrolyte multilayer microcapsules and embedded in a hydrogel matrix. These sensors were fully characterized for pH sensitivity and reproducibility. They were further combined with glucose oxidase (co-encapsulated with the nanoparticles) to generate a glucose-sensitive response, and the Raman signals of the system were recorded in the physiological glucose range (0-400 mg/dL). The results demonstrate SERS based sensing via pH sensitive Raman molecule modified gold nanoparticles (MBA-AuNPs) encapsulated within hydrogels. Further, upon incorporation of GOx, a pH decrease was observed due to glucose oxidization. However, the diffusion-reaction system will need to be better balanced to achieve a more glucose-proportional change for steady-state pH.

SERS active colloidal nanoparticles for the detection of small blood biomarkers using aptamers

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.

Optimization of surface enhanced Raman scattering (SERS) assay for the transition from benchtop to handheld Raman systems

Human biomarkers are indicative of the body’s relative state prior to the onset of disease, and sometimes before symptoms present. While blood biomarker detection has achieved considerable success in laboratory settings, its clinical application is lagging and commercial point-of-care devices are rare. A physician’s ability to detect biomarkers such as microRNA-17, a potential epigenetic indicator of preeclampsia in pregnant woman, could enable early diagnosis and preventive intervention as early as the 1st trimester. One detection approach employing DNA-functionalized nanoparticles to detect microRNA-17, in conjunction with surface-enhanced Raman spectroscopy (SERS), has shown promise but is hindered, in part, by the use of large and expensive benchtop Raman microscopes. However, recent strides have been made in developing portable Raman systems for field applications. Characteristics of the SERS assay responsible for strengthening the assay’s plasmonic response were explored, whilst comparing the results from both benchtop and portable Raman systems. The Raman spectra and intensity of three different types of photoactive molecules were compared as potential Raman reporter molecules: chromophores, fluorophores, and highly polarizable small molecules. Furthermore, the plasmonic characteristics governing the formation of SERS colloidal nanoparticle assemblies in response to DNA/miRNA hybridization were investigated. There were significant variations in the SERS enhancement in response to microRNA-17 using our assay depending on the excitation lasers at wavelengths of 532 nm and 785 nm, depending on which of the three different Raman systems were used (benchtop, portable, and handheld), and depending on which of the three different Raman reporters (chromophore, fluorophore, or Raman active molecule) were used. Analysis of data obtained did indicate that signal enhancement was better for the chromophore (MGITC) and Raman active molecule (DTNB) than it was for the fluorophore (TRITC) and that, although it is possible to obtain enhancements when using excitation lasers that do not directly coincide with the optical properties of the Raman reporter molecule, clearly the enhancements are more significant when it reaches to the characteristic wavelengths of those molecules.

A magneto-fluidic nanoparticle trapping platform for surface-enhanced Raman spectroscopy

A microfluidic device utilizing magnetically activated nickel (Ni) micropads has been developed for controlled localization of plasmonic core-shell magnetic nanoparticles, specifically for surface enhanced Raman spectroscopy (SERS) applications. Magnetic microfluidics allows for automated washing steps, provides a means for easy reagent packaging, allows for chip reusability, and can even be used to facilitate on-chip mixing and filtration towards full automation of biological sample processing and analysis. Milliliter volumes of gold-coated 175-nm silica encapsulated iron oxide nanoparticles were pumped into a microchannel and allowed to magnetically concentrate down into 7.5 nl volumes over nano-thick lithographically defined Ni micropads. This controlled aggregation of core-shell magnetic nanoparticles by an externally applied magnetic field not only enhances the SERS detection limit within the newly defined nanowells but also generates a more uniform (∼92%) distribution of the SERS signal when compared to random mechanical aggregation. The microfluidic flow rate and the direction and strength of the magnetic field determined the overall capture efficiency of the magneto-fluidic nanoparticle trapping platform. It was found that a 5 μl/min flow rate using an attractive magnetic field provided by 1 × 2 cm neodymium permanent magnets could capture over 90% of the magnetic core-shell nanoparticles across five Ni micropads. It was also observed that the intensity of the SERS signal for this setup was 10-fold higher than any other flow rate and magnetic field configurations tested. The magnetic concentration of the ferric core-shell nanoparticles causes the SERS signal to reach the steady state within 30 min can be reversed by simply removing the chip from the magnet housing and sonicating the retained particles from the outlet channel. Additionally, each magneto-fluidic can be reused without noticeable damage to the micropads up to three times.

Aptamer conjugated silver nanoparticles for the detection of interleukin 6

The controlled assembly of plasmonic nanoparticles by a molecular binding event has emerged as a simple yet sensitive methodology for protein detection. Metallic nanoparticles (NPs) coated with functionalized aptamers can be utilized as biosensors by monitoring changes in particle optical properties, such as the LSPR shift and enhancement of the SERS spectra, in the presence of a target protein. Herein we test this method using two modified aptamers selected for the protein biomarker interleukin 6, an indicator of the dengue fever virus and other diseases including certain types of cancers, diabetes, and even arthritis. IL6 works by inducing an immunological response within the body that can be either anti-inflammatory or pro-inflammatory. The results show that the average hydrodynamic diameter of the NPs as measured by Dynamic Light Scattering was ~42 nm. After conjugation of the aptamers, the peak absorbance of the AgNPs shifted from 404 to 408 nm indicating a surface modification of the NPs due to the presence of the aptamer. Lastly, preliminary results were obtained showing an increase in SERS intensity occurs when the IL-6 protein was introduced to the conjugate solution but the assay will still need to be optimized in order for it to be able to monitor varying concentration changes within and across the desired range.