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.
A micro- to nanochannel nanoparticle aggregating device that does not require any input energy to organize the particles to a specific location, i.e., no pumps, plugs, heat, or magnets, has been designed and used to characterize the surface-enhanced Raman spectroscopy (SERS) signal from four unique functionalized nanoparticles (gold, silver-gold nanocages, silver nanocubes, and silica-gold nanoshells). The SERS signal was assessed in terms of the peak signal strength from the four different Raman reporter functionalized nanoparticles to determine which nanoparticle had better utility in the channel to provide the most robust platform for a future biological analyte detection device. The innovation used to fabricate the micro- to nanochannel device is described; the TEM images of the nanoparticles are shown; the absorption data for the nanoparticles are given; and the spectral data for the Raman reporter, mercaptobenzoic acid (MBA), are depicted. In the micro- to nanochannel described in this work, 5 μl of 22.3 μM MBA functionalized silver nanocubes were determined to have the strongest SERS enhancement.
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.
Signal transductions including multiple protein post-translational modifications (PTM), protein-protein interactions (PPI), and protein-nucleic acid interaction (PNI) play critical roles for cell proliferation and differentiation that are directly related to the cancer biology. Traditional methods, like mass spectrometry, immunoprecipitation, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy require a large amount of sample and long processing time. “microchannel for multiple-parameter analysis of proteins in single-complex (mMAPS)”we proposed can reduce the process time and sample volume because this system is composed by microfluidic channels, fluorescence microscopy, and computerized data analysis. In this paper, we will present an automated mMAPS including integrated microfluidic device, automated stage and electrical relay for high-throughput clinical screening. Based on this result, we estimated that this automated detection system will be able to screen approximately 150 patient samples in a 24-hour period, providing a practical application to analyze tissue samples in a clinical setting.
Rapid assessment of radiation exposure to sensitive organs like the gut is extremely important for large populations exposed to ionized radiation, for instance during warfare. Recent results have shown that plasma citrulline levels appear to track gut function after irradiation levels in mice and humans. The current ways to monitor blood citrulline levels are bulky, laborious, time-consuming and expensive methods. Therefore, an optofludic point-of-care (POC) system using surface enhanced Raman spectroscopy to measure plasma citrulline as a marker for radiation exposure that overcomes the above issues is being developed.
As a first step toward development of this system four colloidal nanoparticles, spherical gold, silver cubes, silica-gold nanoshells, and silver-gold nanocages have been analyzed for use in the POC system. Transmission electron microscopy (TEM) images have been taken of each nanoparticle to visualize the morphology of the nanoparticles, which is vital for SERS. Ultraviolet-visible (UV/Vis) spectroscopy was also collected to verify the extinction spectra for each nanoparticle was in resonance with the excitation wavelength. The nanoparticles were functionalized with mercaptobenzoic acid (MBA), a Raman reporter molecule, and SERS spectra were collected to determine which has better utility in a novel micro-to-nanochannel. The data showed that the silver nanocubes have a larger enhancement factor than the gold nanospheres, nanoshells, or nanocages. Currently, these nanocubes are being functionalized with the citulline for assessing the concentration sensitivity and dynamic range for ultimate use as a marker for radiation.
In this presentation we will discuss the development of a point-of-care optofluidic device that uses gold nanoparticlebased
surface enhanced Raman spectroscopy (SERS) for detection of blood biomarkers. SERS approaches have been
successfully used for detection of analytes due to the large enhancements provided by the interaction between the light,
gold particles, and analyte. However, SERS approaches developed for use to accurately quantify an analyte have
suffered from a lack of repeatability. We will describe our SERS optofluidic device with functionalized nanoparticles
that helps to overcome these problems and will show results with a focus on blood cardiac biomarkers.
The overall goal of this research is to develop a new point-of-care system for early detection and
characterization of cardiac markers to aid in diagnosis of acute coronary syndrome. The envisioned final technology
platform incorporates functionalized gold colloidal nanoparticles trapped at the entrance to a nanofluidic device
providing a robust means for analyte detection at trace levels using surface enhanced Raman spectroscopy (SERS).
To discriminate a specific biomarker, we designed an assay format analogous to a competitive ELISA. Notably, the
biomarker would be captured by an antibody and in turn displace a peptide fragment, containing the binding epitope
of the antibody labeled with a Raman reporter molecule that would not interfere with blood serum proteins. To
demonstrate the feasibility of this approach, we used C-reactive protein (CRP) as a surrogate biomarker. We
functionalized agarose beads with anti-CRP that were placed outside the nanochannel, then added either
Rhodamine-6-G (R6G) labeled-CRP and gold (as a surrogate of a sample without analyte present), or R6G labeled
CRP, gold, and unlabeled CRP (as a surrogate of a sample with analyte present). Analyzing the spectra we see an
increase in peak intensity in the presence of analyte at characteristic peaks for R6G specifically, 1284 and1567 cm-
1. Further, our results illustrate the reproducibility of the Raman spectra collected for R6G-labeled CRP in the
nanochannel. Overall, we believe that this method will provide the advantage of sensitivity and narrow line widths
characteristic of SERS as well as the specificity toward the biomarker of interest.
A microfluidic single molecule fluorescence-based detection scheme is developed to identify target protein direct from
cell lysate by using polyclonal antibody. Relative concentration of target protein in solution is determined by twodimensional
(2D) photon burst analysis. Compared to conventional ensemble measurement assays, this microfluidic
single molecule approach combines the advantages of higher sensitivity, fast processing time, small sample consumption
and high resolution quantitative analysis.
According to the World Health Organization, cardiovascular disease is the most common cause of death in the world. In
the US, over 115 million people visit the emergency department (ED), 5 million of which may have acute coronary
syndrome (ACS). Cardiac biomarkers can provide early identification and diagnosis of ACS, and can provide
information on the prognosis of the patient by assessing the risk of death. In addition, the biomarkers can serve as criteria
for admission, indicate possibility of re-infarction, or eliminate ACS as a diagnosis altogether. We propose a SERSbased
multi-marker approach towards a point-of-care diagnostic system for ACS. Using a nanofluidic device consisting
of a microchannel leading into a nanochannel, we formed SERS active sites by mechanically aggregating gold particles
(60 nm) at the entrance to the nanochannel (40nm×1μm). The induced capillary flow produces a high density of
aggregated nanoparticles at this precise region, creating areas with enhanced electromagnetic fields within the
aggregates, shifting the plasmon resonance to the near infrared region, in resonance with incident laser wavelength. With
this robust sensing platform, we were able to obtain qualitative information of brain natriuretic peptide (biomarker of
ventricular dysfunction or pulmonary stress), troponin I (biomarker of myocardial necrosis), and C-reactive protein
(biomarker of inflammation potentially caused by atherosclerosis).
Alzheimer's disease (AD), a neurodegenerative disease and the most common cause of dementia, affects 4.5 million
people according to the 2000 US census and is expected to triple to 13.2 million by the year 2050. Since no definitive
pre-mortem tests exist to distinguish AD from mild cognitive impairment due to the natural aging process, we focus on
detecting the beta amyloid (Aβ) protein, the primary component of the senile plaques characteristic of AD. We
specifically detect cytotoxic species of Aβ by exploiting surface enhanced Raman scattering (SERS). Using a
nanofluidic device with a bottleneck shape (a microchannel leading into a nanochannel); we trapped gold colloid
particles (60 nm) at the entrance to the nanochannel, with Aβ restricted within the interstices between the aggregated
nanoparticles. The continuous flow generated from pumping the solution into the device produced size-dependent
trapping of the gold colloid particles, resulting in a high density of aggregated nanoparticles at this precise region,
creating localized "hot spots" in the interstitial region between nanoparticles, and shifting the plasmon resonance to the
near infrared region, in resonance with incident laser wavelength. With this robust sensing platform, we were able to
obtain concentration-dependent SERS spectra of Aβ and of different proteins present in the cerebrospinal fluid of
healthy people and people with Alzheimer's disease.
The Raman scattering signature of molecules has been demonstrated to be greatly enhanced, on the order of 106-1012
times, on roughened metal surfaces and clustered structures such as aggregated colloidal gold. Here we describe a
method that improves reproducibility and sensitivity of the substrate for surface enhanced Raman spectroscopy (SERS)
by using a nanofluidic trapping device. This nanofluidic device has a bottle neck shape composed of a microchannel
leading into a nano channel that causes size-dependent trapping of nanoparticles. The analyte and Au nanoparticles, 60
nm in diameter, in aqueous solution was pumped into the channel. The nanoparticles which were larger than the narrow
channel are trapped at the edge of the channel to render an enhancement of the Raman signal. We have demonstrated
that the Raman scattering signal enhancement on a nanochannel-based colloidal gold cluster is able to detect 10 pM of
adenine, the test analyte, without chemical modification. The efficiency and robustness of the device suggests potential
for single molecule detection and multicomponent detection for biological applications and/or biotoxins.
We have used an embossed plastic microfluidic system for the electrophoretic separation of relatively small molecules followed by electro spray ionization of the analytes. Th separation of dyes has been also visualized in microfluidic systems. A lithographically produced silicon master was used to emboss channels in ZEONOR 1020R plastic. An oxygen plasma was used to convert the plastic channel surface from hydrophobic to hydrophilic characteristics for the separation of molecules in aqueous solution.
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