Final Report: A Nanocrystal Biosensor Array for Simultaneous Detection of Multiple Waterborne Pathogens

EPA Contract Number: EPD05028
Title: A Nanocrystal Biosensor Array for Simultaneous Detection of Multiple Waterborne Pathogens
Investigators: Liu, Yongcheng
Small Business: Nanomaterials & Nanofabrication Laboratories
EPA Contact: Manager, SBIR Program
Phase: I
Project Period: March 1, 2005 through August 31, 2005
Project Amount: $70,000
RFA: Small Business Innovation Research (SBIR) - Phase I (2005) RFA Text |  Recipients Lists
Research Category: SBIR - Nanotechnology , Small Business Innovation Research (SBIR) , Nanotechnology

Description:

The goal of this research project was to develop a new generation of sensitive, rapid, portable, robust, and inexpensive biosensors for simultaneous detection of multiple waterborne pathogens in water products and the environment. In Phase I, Nanomaterials and Nanofabrication Laboratories (NN-Labs) proposed to demonstrate the feasibility of the biosensor technology by focusing on detection of one waterborne pathogen (e.g., Escherichia coli O157:H7). The research plan was to conjugate one color nanocrystal onto the antibody as a biomarker, immobilize the antibody without the nanocrystal marker onto the porous membrane surface, design a cartridge for constructing a new flow cell setup to specifically capture and concentrate the waterborne pathogens, and compare the results with enzyme-linked immunosorbent technology.

Summary/Accomplishments (Outputs/Outcomes):

NN-Labs developed a universal, reliable, and inexpensive protocol for modifying highly luminescent semiconductor nanocrystals. This technology enabled the firm to make three different types of dendron ligand-coated nanocrystals (dendron-nanocrystals) that are ready to conjugate onto various antibodies (see Figure 1). The dendron-nanocrystals retain the excellent optical properties of the inorganic core, CdSe/ZnS core/shell nanocrystals, which will be used as biomarkers for labeling the antibodies in the Phase II project. Dendron-nanocrystals under ultraviolet (UV) radiation are shown in Figure 1. The orange nanocrystal has been used to label two types of antibodies: (1) anti-hepatitis B antibody, and (2) anti-E. coli O157:H7 antibody.

The Water-Soluble and Biocompatible Cdse/Zns Core-Shell Dendron-Nanocrystals Present the Same Emission Colors as the Initial Ones in Hexane Solution

Figure 1. The Water-Soluble and Biocompatible Cdse/Zns Core-Shell Dendron-Nanocrystals Present the Same Emission Colors as the Initial Ones in Hexane Solution

NN-Labs developed a novel protocol for immobilizing various antibodies onto a porous membrane surface and has immobilized two kinds of antibodies onto the surface of the porous polymer membranes by covalent binding: (1) anti-hepatitis B surface Ag (HBsAg), antibody, and (2) anti-E. coli O157:H7 antibody. The immobilized antibodies retain their activities and can capture the corresponding antigens. The antibody-immobilized membranes have been used to detect the corresponding antigens.

NN-Labs also developed a universal, reliable, simple, and inexpensive protocol for conjugating nanocrystals onto various antibodies and has made two types of orange nanocrystal-conjugated antibodies: (1) orange nanocrystal-conjugated anti-HBsAg antibody, and (2) orange nanocrystal-conjugated anti-E. coli O157:H7 antibody. These orange nanocrystal-labeled antibodies retain both the nanocrystal optical properties as biomarkers and the biological activities of the antibodies to form immunocomplexes with the corresponding antigens. These nanocrystal-labeled antibodies have been used to detect the corresponding antigens.

NN-Labs has constructed A cartridge for the new flow cell setup has been constructed to detect various pathogens (see Figure 2).

Figure 2. The Cartridge for the Flow Cell System

NN-Labs demonstrated that this biosensor technology can detect hepatitis B surface Ag, a type of virus. The typical relationship between the photoluminescence and the antigen concentration is presented in Figure 3.

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Figure 3. Concentration Curve of HbsagVersusPhotoluminescence Signal

As shown in this figure, the peak intensity/area increases with the concentration of HBsAgsolution. The detection limit obtained in this study is approximately 5 ng/mL. The assay time is approximately 30 minutes.

NN-Labs also demonstrated that this biosensor technology can detect E. coli O157:H7, a typical waterborne pathogen. The calibration curve for the detection of E. coli is illustrated in Figure 4. As shown in this figure, the peak intensity/area increases with E. coli O157:H7 concentration. The detection limit obtained in this study is obviously approximately 2.3 x 100 CFU/mL. It implies that the technology can detect as low as 1 CFU/mL of E. coli O157:H7 culture without any enrichment or incubation in a 30-minute assay time. Its working range is very broad—from 2.3 x100 to 2.3 x107 CFU/mL.

Concentration curve of E.  coli O157:H7versusphotoluminescence signal.

Figure 4. Concentration curve of E. coli O157:H7versusphotoluminescence signal.

The selectivity of the biosensor technology is good, and results are presented in Figure 5. Very little interference was observed from the other pathogens, which included Salmonella typhimurium, and Listeria monocytogenes.

Figure 5. The Selectivity of The Biosensor Technology. Sample A: 1.2 percent BSA; Sample B: the mixture of 5.1 x105 CFU/mLof L. monocytogenes and1.2 x105 CFU/mLof S. typhimurium;Sample C: 4.1 x104 CFU/mLof E. coli O157:H7; Sample D: the mixture of 5.1 x105 CFU/mLof L. monocytogenes, 1.2 x105 CFU/mL of S. typhimurium and4.1 x104 CFU/mL of E. coli O157:H7. Note: The E. coli concentration was chosen to be 10 times lower than that of the other types of bacteria.

Conclusions:

NN-Labs successfully developed universal, reliable, simple, and inexpensive protocols for modifying orange-colored nanocrystals with dendron ligands to become water- soluble/biocompatible, for conjugating nanocrystals onto various antibodies, and for immobilizing various antibodies onto the membrane surface. The nanocrystal biosensor technology, in combination with an antibody-immobilized membrane, has been demonstrated as a sensitive, rapid, portable, robust, and inexpensive biosensor for the detection of waterborne pathogens. The detection limit is as low as 1 CFU/mL, without any incubation and enrichment. The detection time could be shortened to 30 minutes. The working range is very broad, from 100 to 107 CFU/mL. The technology is feasible for simultaneous detection of multiple waterborne pathogens.

A manuscript will be submitted to a peer-reviewed journal, such as Nature Biotechnology or Analytical Chemistry. A patent is being considered.

Supplemental Keywords:

water, nanocrystals, dendron ligand-coated nanocrystals, biosensor, biosensor array, Escherichia coli O157:H7, porous membrane, antibodies, immobilized antibodies, antigen, anti-hepatitis B surface Ag, antibody, nanocrystal conjugates, flow cell, photoluminescence, waterborne pathogens, Salmonella typhimurium, Listeria monocytogenes, EPA, small business, SBIR,, RFA, Scientific Discipline, INTERNATIONAL COOPERATION, Water, TREATMENT/CONTROL, Ecosystem Protection/Environmental Exposure & Risk, Sustainable Industry/Business, Chemical Engineering, Environmental Chemistry, Technology, Monitoring/Modeling, Environmental Monitoring, New/Innovative technologies, Drinking Water, Engineering, Chemistry, & Physics, Environmental Engineering, monitoring, nanosensors, homeland security, environmental measurement, detection, field portable systems, field portable monitoring, biopollution, drinking water regulations, nanotechnology, waterborne pathogens, community water system, field monitoring, chemical detection techniques, biotechnology, analytical chemistry, environmental contaminants, analytical methods, microbial risk management, fluorescent nanoparticle aptamer magnetic bead sensor, measurement, resonating microsensor, drinking water contaminants, biosensors, biosensor, drinking water system