Lateral flow immunoassays are paper-based tests that can be used to detect different pathogenic biomarkers at the point-of-use. Conventionally, detection antibodies labelled with gold nanoparticles form sandwich immunoassays between the target antigen and capture antibody on the test line/spot when the biomarker is present in a sample. They provide rapid, visual and yes/no answers based on the appearance of the gold nanoparticles on the strip. However, lateral flow tests suffer from poor sensitivity which can lead to false negative results when the biomarker concentration is below the visual threshold of the test, especially at early infection stages. They also can’t accurately quantify the biomarker concentration which is important in monitoring the progression of an infection or the effectiveness of a treatment. Accordingly, lateral flow test should be combined with a read-out system that is convenient for points of use and could enable the accurate, sensitive and rapid quantification of the test results. In this work, we discuss the integration of surface enhanced Raman scattering technique with the lateral flow test in one platform to improve the test sensitivity and quantification capability, while maintaining the user-friendly and point-of-use features the lateral flow test provides. We have applied this approach for the detection of Clostridioides difficile and SARS-COV-2, as demonstrators for pathogenic infections, using new recognition elements in the test as a proof-of-concept.
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.
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.
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.
We report a versatile nanophotonic biosensing platform that enables both colorimetric detection and enhanced Raman spectroscopy detection of molecular binding events. Through the integration of electron-beam lithography, dip-pennanolithography and molecular self-assembly, we demonstrate plasmonic nanostructures which change geometry and plasmonic properties in response to molecularly-mediated nanoparticle binding events. These biologically-active nanostructured surfaces hold considerable potential for use as multiplexed sensor platforms for point-of-care diagnostics, and as scaffolds for a new generation of molecularly dynamic metamaterials.
KEYWORDS: Glucose, Luminescence, Sensors, Proteins, Energy transfer, Blood, Atrial fibrillation, Fluorescence resonance energy transfer, Chemistry, Resonance energy transfer
Fluorescent glucose sensing technologies have been identified as possible alternatives to current continuous glucose monitoring approaches. We have recently introduced a new, smart fluorescent ligand to overcome the traditional problems of ConA-based glucose sensors. For this assay to be translated into a continuous glucose monitoring device where both components are free in solution, the molecular weight of the smart fluorescent ligand must be increased. We have identified ovalbumin as a naturally-occurring glycoprotein that could serve as the core-component of a 2nd generation smart fluorescent ligand. It has a single asparagine residue that is capable of displaying an N-linked glycan and a similar isoelectric point to ConA. Thus, binding between ConA and ovalbumin can potentially be monovalent and sugar specific. This work is the preliminary implementation of fluorescently-labeled ovalbumin in the ConA-based assay. We conjugate the red-emitting, long-lifetime azadioxatriangulenium (ADOTA+) dye to ovalbumin, as ADOTA have many advantageous properties to track the equilibrium binding of the assay. The ADOTA-labeled ovalbumin is paired with Alexa Fluor 647-labeled ConA to create a Förster Resonance Energy Transfer (FRET) assay that is glucose dependent. The assay responds across the physiologically relevant glucose range (0-500 mg/dL) with increasing intensity from the ADOTA-ovalbumin, showing that the strategy may allow for the translation of the smart fluorescent ligand concept into a continuous glucose monitoring device.
The unique optical properties associated with nanostructured materials that support the excitation of surface plasmons
offer many new opportunities for the enhanced optical investigation of biological materials that pose a security threat. In
particular, ricin is considered a significant bioterrorism risk due to its high toxicity combined with its ready availability
as a byproduct in castor oil production. Therefore, the development of optical techniques capable of rapid on-site toxin
detection with high molecular specificity and sensitivity continues to be of significant importance. Furthermore,
understanding of the ricin cell entry and intracellular pathways remains poor due to a lack of suitable bioanalytical
techniques. Initial work aimed at simultaneously tackling both these issues is described where different approaches for
the nanoparticle labeling of ricin are investigated along with changes in ricin toxicity associated with the labeling
process.
There is a need for fast, reliable and sensitive biosensor arrays. We have used nanostructured plasmonic gold surfaces for
the detection of biological species by surface enhanced resonance Raman scattering (SERRS). Careful, directed
placement by Dip-Pen Nanolithography (DPN) of the biological species or capture chemistry, within the array facilitates
efficient read out via fast Raman line mapping. In addition, we can apply parallel deposition methods to enhance the
throughput of these combined techniques. SERRS is an extremely sensitive spectroscopic technique that offers several
advantages over conventional fluorescence detection. For example, the high sensitivity of the method allows detection of
DNA capture from single plasmonic array "pixels" ~1 μm2 in area. Additionally, the information rich nature of the
SERRS spectrum allows multiple levels of detection to be embedded into each pixel, further increasing the information
depth of the array. By moving from micro- to nano-scale features, sensor chips can contain up to 105 times more
information, dramatically increasing the capacity for disease screening.
Surface enhanced resonance Raman scattering (SERRS) is an analytical technique with several advantages over
competitive techniques in terms of improved sensitivity and multiplexing. We have made great progress in the
development of SERRS as a quantitative analytical method, in particular for the detection of DNA. SERRS is an
extremely sensitive and selective technique which when applied to the detection of labelled DNA sequences allows
detection limits to be obtained which rival, and in most cases, are better than fluorescence. Here the conditions are
explored which will enable the successful detection of DNA using SERRS. The enhancing surface which is used is
crucial and in this case suspensions of nanoparticles were used as they allow quantitative behaviour to be achieved and
allow analogous systems to current fluorescence based systems to be made. The aggregation conditions required to
obtain SERRS of DNA are crucial and herein we describe the use of spermine as an aggregating agent. The nature of the
label which is used, be it fluorescent, positively or negatively charged also effects the SERRS response and these
conditions are again explored here. We have clearly demonstrated the ability to identify the components of a mixture of 5
analytes in solution by using two different excitation wavelengths and also of a 6-plex using data analysis techniques.
These conditions will allow the use of SERRS for the detection of target DNA in a meaningful diagnostic assay.
Functionalised nanoparticles have been used in a number of studies including detection of DNA at ultra low
concentrations, immuno-histochemistry and more recently as substrates for surface enhanced resonance Raman
scattering (SERRS) based imaging approaches. The advantages of using metallic nanoparticles are that they are
very bright in terms of their optical characteristics and also can be functionalised to provide a SERRS response and
hence provide a unique Raman fingerprint. Here we present the functionalisation of gold and silver nanoparticles in
such a way that the enhancement effect can be greatly increased through biological interaction and as such
effectively turn on the SERRS effect. In an advancement of this nanoparticles have been used as imaging agents for
single cells when functionalised with an appropriate antibody and can give information on the expression of specific
receptors on cell surfaces as well as sub-cellular compartmentalisation information.
Gold and silver nanoparticles functionalized with oligonucleotides can be used for the detection of specific
sequences of DNA. We show that gold nanoparticles modified with locked nucleic acid (LNA) form stronger
duplexes with a single stranded DNA target and offer better discrimination against single base pair mismatches
than analogous DNA probes. Our LNA nanoparticle probes have also been used to detect double stranded DNA
through triplex formation, whilst still maintaining selectivity for only complementary targets. Nanoparticle
conjugates embedded with suitable surface enhanced resonance Raman scattering (SERRS) labels have been
synthesized enabling simultaneous detection and identification of multiple DNA targets.
There is a growing need for fast reliable biosensor arrays for disease screening. We have used nanostructured plasmonic gold
surfaces for the detection of biological species by surface enhanced resonance Raman scattering (SERRS). Careful, directed
placement by Dip-pen Nanolithography (DPN) of the biological species or capture chemistry, within the array facilitates
efficient read out via ultra fast Raman line mapping. Further, we can transition the serial placement of biological species /
capture chemistry to a massively parallel deposition method, and this flexibility is key to enhancing the throughput of these
combined techniques by many orders of magnitude. SERRS is an extremely sensitive spectroscopic technique that offers
several advantages over conventional fluorescence detection. For example, the high sensitivity of the method allows
detection of DNA capture from single plasmonic array "pixels" ~1 μm2 in area. Additionally, the information rich nature of
the SERRS spectrum allows multiple levels of detection to be embedded into each pixel, further increasing the information
depth of the array. By moving from micro- to nano-scale features, sensor chips can contain up to 105 times more information,
dramatically increasing the capacity for disease screening.
KEYWORDS: Fluorescence resonance energy transfer, Luminescence, Systems modeling, Molecules, Cobalt, Ions, Time resolved spectroscopy, Microelectromechanical systems, Molecular energy transfer, Spectroscopy
We demonstrate a new fluorescence resonance energy transfer (FRET) based approach to determine the donor-acceptor
distributions and apply it to two model molecular systems: double stranded DNA labeled with Hoechst 33258 and FAM, and
perylene randomly surrounded by cobalt ions in a bulk solution. The approach makes some generic assumptions regarding
the FRET kinetics, but no a priori assumptions regarding the distribution function.
Surface enhanced resonance Raman scattering (SERRS) provides an increase in sensitivity over Raman scattering of
about 1014 and rivals fluorescence in terms of its quantum efficiency. With the use of modern edge and notch filters and
CCD cameras, the price and complexity of Raman spectroscopy equipment has decreased rapidly. This means that the
potential advantages of SERRS are now much easier to release for use for practical purposes. The technique has
specific advantages in terms of sensitivity and coding for use for tagging.
Surface-enhanced resonance Raman scattering (SERRS) is a very sensitive and selective detection method that can be used for the analysis of both DNA and P-450s. A number of factors have limited the broader application of the technique. These limitations are described and addressed. An approach to reduce the problems associated with variation of the silver colloids used to provide surface enhancement and chemical methodologies that ensure surface adsorption are presented. A practical approach was used to investigate the nature of the effect. This approach has highlighted the importance of resonance enhancement for ultimate sensitivity. Two approaches to achieve successful detection of DNA using SERRS are described, and, using these two approaches, the possibility of multiplexing is also demonstrated. The analysis of proteins by SERRS is discussed and P-450 is presented as a specific example of the information that may be gained from SERRS of proteins.
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