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Single step nanoplasmonic immunoassay for the measurement of protein biomarkers.

Prabhulkar S, de la Zerda A, Paranjape A, Awdeh RM - Biosensors (Basel) (2013)

Bottom Line: This nanoplasmonic immunoassay can act as a simple, selective, sensitive strategy for effective disease diagnosis.It offers advantages such as wide detection range, increased speed of analysis (due to fewer incubation/washing steps), and no label development as compared to traditional immunoassay techniques.Our future goal is to incorporate this detection strategy onto a microfluidic platform to be used as a point-of-care diagnostic tool.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Miami-Bascom Palmer Eye Institute, Miami, FL 33136, USA; E-Mails: sprabhulkar@med.miami.edu (S.P.); asparanjape@gmail.com (A.P.).

ABSTRACT
A nanoplasmonic biosensor for highly-sensitive, single-step detection of protein biomarkers is presented. The principle is based on the utilization of the optical scattering properties of gold nanorods (GNRs) conjugated to bio-recognition molecules. The nanoplasmonic properties of the GNRs were utilized to detect proteins using near-infrared light interferometry. We show that the antibody-conjugated GNRs can specifically bind to our model analyte, Glucose Transporter-1 (Glut-1). The signal intensity of back-scattered light from the GNRs bound after incubation, correlated well to the Glut-1 concentration as per the calibration curve. The detection range using this nanoplasmonic immunoassay ranges from 10 ng/mL to 1 ug/mL for Glut-1. The minimal detectable concentration based on the lowest discernable concentration from zero is 10 ng/mL. This nanoplasmonic immunoassay can act as a simple, selective, sensitive strategy for effective disease diagnosis. It offers advantages such as wide detection range, increased speed of analysis (due to fewer incubation/washing steps), and no label development as compared to traditional immunoassay techniques. Our future goal is to incorporate this detection strategy onto a microfluidic platform to be used as a point-of-care diagnostic tool.

No MeSH data available.


Calibration curve depicting averaged OCT signal intensity versus corresponding initial concentration of Glut-1 protein.
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biosensors-03-00077-f003: Calibration curve depicting averaged OCT signal intensity versus corresponding initial concentration of Glut-1 protein.

Mentions: After optimizing the key steps in the molecular architecture of the sensor, we assessed the performance of the immunosensor by challenging it with increasing concentrations of Glut-1 protein from 0.1 ng/mL to 1 mg/mL. The OCT images of the sonicated 96-well plates containing an initial Glut-1 concentration of 0.1 ng/mL, 1 ng/mL, 10 ng/mL, 50 ng/mL, 100 ng/mL, 250 ng/mL, 500 ng/mL, 750 ng/mL, and 1 mg/mL are shown in Figure 2, PBS buffer acts as the negative control. A thorough evaluation of Figure 2, can allow us to discern that the scattering visible in the OCT image due to the presence of GNRs intensifies in proportion to the concentration of Glut-1. The OCT images were then normalized and, by choosing a region of interest, the average OCT signal intensity was calculated for each image. A calibration curve was plotted using the average OCT signal intensity (four replicates × three distinct plates = 12 data points/Glut-1 concentration) as shown in Figure 3. Based on the calibration curve we can conclude that the nano-optical protein measurement sensing strategy developed is capable of detection Glut-1, within a detection range of 10 ng/mL to 1µg/mL of Glut-1. The sensitivity of this assay, or lower limit of detection was defined as the lowest protein concentration that could be differentiated from the negative control (PBS buffer). The sensitivity of the assay was measured to be 10 ng/mL. The nano optical sensing strategy described provides a wide detection range as compared to the commercially available Glut-1 ELISA kits which perform within a range of 0.312 ng/mL–20 ng/mL (USCN Life Science Inc.), 1.15 ng/mL–20 ng/mL (Labome) and 1.15 ng/mL–20 ng/mL (Cusabio Biotech Co., Ltd.). Spherical gold nanoparticles have also been used for the development of similar biosensors based on the principles of exploiting the plasmonic properties of gold. Maier et al., describe the development of an optical immunochip based sensor for the detection allergens in food matrices using spherical gold nanoparticles. The single step immunoassay displays a 10 ng/ml detection limit for the measurement of ovalbumin [19]. Thanh et al. developed an aggregation based immunoassay for anti-Protein A using spherical gold nanoparticles which displays a dynamic range of two orders of magnitude and a limit of detection of 1 μg/mL [16].


Single step nanoplasmonic immunoassay for the measurement of protein biomarkers.

Prabhulkar S, de la Zerda A, Paranjape A, Awdeh RM - Biosensors (Basel) (2013)

Calibration curve depicting averaged OCT signal intensity versus corresponding initial concentration of Glut-1 protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263591&req=5

biosensors-03-00077-f003: Calibration curve depicting averaged OCT signal intensity versus corresponding initial concentration of Glut-1 protein.
Mentions: After optimizing the key steps in the molecular architecture of the sensor, we assessed the performance of the immunosensor by challenging it with increasing concentrations of Glut-1 protein from 0.1 ng/mL to 1 mg/mL. The OCT images of the sonicated 96-well plates containing an initial Glut-1 concentration of 0.1 ng/mL, 1 ng/mL, 10 ng/mL, 50 ng/mL, 100 ng/mL, 250 ng/mL, 500 ng/mL, 750 ng/mL, and 1 mg/mL are shown in Figure 2, PBS buffer acts as the negative control. A thorough evaluation of Figure 2, can allow us to discern that the scattering visible in the OCT image due to the presence of GNRs intensifies in proportion to the concentration of Glut-1. The OCT images were then normalized and, by choosing a region of interest, the average OCT signal intensity was calculated for each image. A calibration curve was plotted using the average OCT signal intensity (four replicates × three distinct plates = 12 data points/Glut-1 concentration) as shown in Figure 3. Based on the calibration curve we can conclude that the nano-optical protein measurement sensing strategy developed is capable of detection Glut-1, within a detection range of 10 ng/mL to 1µg/mL of Glut-1. The sensitivity of this assay, or lower limit of detection was defined as the lowest protein concentration that could be differentiated from the negative control (PBS buffer). The sensitivity of the assay was measured to be 10 ng/mL. The nano optical sensing strategy described provides a wide detection range as compared to the commercially available Glut-1 ELISA kits which perform within a range of 0.312 ng/mL–20 ng/mL (USCN Life Science Inc.), 1.15 ng/mL–20 ng/mL (Labome) and 1.15 ng/mL–20 ng/mL (Cusabio Biotech Co., Ltd.). Spherical gold nanoparticles have also been used for the development of similar biosensors based on the principles of exploiting the plasmonic properties of gold. Maier et al., describe the development of an optical immunochip based sensor for the detection allergens in food matrices using spherical gold nanoparticles. The single step immunoassay displays a 10 ng/ml detection limit for the measurement of ovalbumin [19]. Thanh et al. developed an aggregation based immunoassay for anti-Protein A using spherical gold nanoparticles which displays a dynamic range of two orders of magnitude and a limit of detection of 1 μg/mL [16].

Bottom Line: This nanoplasmonic immunoassay can act as a simple, selective, sensitive strategy for effective disease diagnosis.It offers advantages such as wide detection range, increased speed of analysis (due to fewer incubation/washing steps), and no label development as compared to traditional immunoassay techniques.Our future goal is to incorporate this detection strategy onto a microfluidic platform to be used as a point-of-care diagnostic tool.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Miami-Bascom Palmer Eye Institute, Miami, FL 33136, USA; E-Mails: sprabhulkar@med.miami.edu (S.P.); asparanjape@gmail.com (A.P.).

ABSTRACT
A nanoplasmonic biosensor for highly-sensitive, single-step detection of protein biomarkers is presented. The principle is based on the utilization of the optical scattering properties of gold nanorods (GNRs) conjugated to bio-recognition molecules. The nanoplasmonic properties of the GNRs were utilized to detect proteins using near-infrared light interferometry. We show that the antibody-conjugated GNRs can specifically bind to our model analyte, Glucose Transporter-1 (Glut-1). The signal intensity of back-scattered light from the GNRs bound after incubation, correlated well to the Glut-1 concentration as per the calibration curve. The detection range using this nanoplasmonic immunoassay ranges from 10 ng/mL to 1 ug/mL for Glut-1. The minimal detectable concentration based on the lowest discernable concentration from zero is 10 ng/mL. This nanoplasmonic immunoassay can act as a simple, selective, sensitive strategy for effective disease diagnosis. It offers advantages such as wide detection range, increased speed of analysis (due to fewer incubation/washing steps), and no label development as compared to traditional immunoassay techniques. Our future goal is to incorporate this detection strategy onto a microfluidic platform to be used as a point-of-care diagnostic tool.

No MeSH data available.