Final Report: MEMS Biosensor for In Situ Drinking Water Analysis

EPA Contract Number: 68D99052
Title: MEMS Biosensor for In Situ Drinking Water Analysis
Investigators: Salazar, Noe
Small Business: JCP Technologies Inc.
EPA Contact:
Phase: I
Project Period: September 1, 1999 through March 1, 2000
Project Amount: $70,000
RFA: Small Business Innovation Research (SBIR) - Phase I (1999) RFA Text |  Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , SBIR - Monitoring , Small Business Innovation Research (SBIR)

Description:

The goal of this Phase I program was to demonstrate proof-of-concept for a low-cost and easy to use, highly sensitive and specific biosensor for the detection of pathogenic microorganisms in drinking water systems. Due to EPA and CDC priorities, Cryptosporidium parvum was targeted. The technical objectives were:

Develop a design for and demonstrate microfabrication of the steps required for sample isolation, cleanup and separation, as well as DNA detection;

Develop the branch DNA assay with target capture, detection and signal amplification for C. parvum oocysts; and

Determine a sensitive signal transduction method and configure an ?on-chip' assay format for detection of oocysts to be used in conjunction with the chip.

Summary/Accomplishments (Outputs/Outcomes):

The bulk of the research focused on the first two objectives. The first effort centered on the design of an integrated system to perform the various steps required for sample processing and target detection. Completing the system design, the required components were identified and it was evident that the key necessary component not sufficiently developed was the thermal cycler. The microfabrication effort then focused on the development of this heater module.

In this task, a mask for a microchip thermal cycler with both the heater component and a temperature sensor for feedback was designed and fabricated. Several wafers with thermal cyclers of varying dimensions and with varying resistance characteristics were microfabricated at the Washington Technology Center. Test and control circuitry was built and these heater modules were tested with fluid for performance. A number of chips successfully demonstrated the desired performance characteristics. This highly successful focus both completed the necessary component inventory, and also demonstrated the microfabrication techniques that can be employed in the modification or further development of other components.

The second critical task was the development of the assay for capture, lysis, and detection of Cryptosporidium oocyst DNA. This effort was broken down into 3 major tasks: design and fabrication of the necessary oligonucleotides for the bDNA assay; obtaining C. parvum oocysts and DNA for testing; and demonstrating proof-of-concept for the assay. In this effort, the sequences for the oligonucleotides required as the various components of the bDNA assay against C. parvum were designed and fabricated. The difficult task of obtaining Cryptosporidium DNA from oocysts was successfully completed, and tests were conducted to demonstrate the assay.

The assay is dependent on three critical components: the capture probes, the detection or preamplifier probes, and the universal reagents which act as signal amplifiers. Universal reagents are not specific to the target being tested for and have been proven effective many times by numerous researchers. Our efforts therefore centered on proving that the specific DNA probes that JCP designed for capture and detection of Cryptosporidium oocyst wall protein DNA would work. In repeated tests, the assay was an unqualified success.

The final effort was investigating the means to transpose the assay developed into a system using the microfabricated components for a compact, low-cost design and determining the signal transduction method to be used. Our investigation concluded that a fluorescence detection methodology would be optimal. In the process of developing an advanced design for Phase II implementation, we also set upon a modular approach for integration of the various components required. In related work, JCP has developed a modular microfluidic interconnect technology which will be ideal in this application.

Conclusions:

JCP Technologies was extremely successful in demonstrating the application of a highly specific and sensitive branch DNA assay to the detection of Cryptosporidium parvum oocysts. While the assay has been shown as a very effective means of detection for other organisms, this is the first time it has been applied to this critical pathogen. This success includes the development of the proper sequence for the oligonucleotide DNA probes for use in this detection assay. JCP was also successful in the development of the necessary components and microfabrication process for fabrication of an integrated, compact and low-cost analytical instrument for use in drinking water systems.

In successfully completing the objectives of this Phase I program, JCP Technologies has provided proof-of-concept for a novel, highly specific and sensitive instrument for field use in the detection of Cryptosporidium and other pathogens in drinking water systems. This device has the potential for detection of a broad range of bacterial and viral pathogens in a number of different media including blood samples. The results of a Commercial Assessment Study conducted by Foresight S&T found this technology to be highly commercial. Foresight states: "We believe that its convenience (MEMS technology), speed (results in an hour), and reliability should present clear advantages against other methods already in use or in development." Further, "We believe that, by the end of R&D, [JCP's] technology should be able to meet all the requirements provided by end-users." And finally, "We believe this technology CAN be commercialized." To test their conclusion, Foresight polled three experts in the field on the potential for commercialization of this technology. On a scale of 0 (none) to 10 (certain) the results were 8, 8, and 9 with comments such as: "Technology like this one is highly desirable."

Our Commercialization Plan details our entry strategy to the $300 million potential market for these diagnostic instruments. JCP Technologies is now poised to develop a working prototype in the Phase II of this SBIR and to prepare for commercialization in the Option program.

Supplemental Keywords:

Drinking water analysis, Cryptosporidium; branch DNA; DNA probes; MEMS; biosensor; pathogen detection., RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Microbiology, Monitoring/Modeling, Drinking Water, Engineering, Engineering, Chemistry, & Physics, biosensing, monitoring, pathogens, assays, bacteria, detection, pathogenic microbes, exposure and effects, exposure, bacteria monitoring, detect, in situ sensor, nucleic acid-based detection technology, analyzer, biosensing system, treatment, microbial risk management, measurement, cost-effective sensors, drinking water contaminants, biosensor

SBIR Phase II:

MEMS Biosensor for In Situ Drinking Water Analysis