Final Report: Biosensor design for infectious, water-borne agents

EPA Grant Number: SU836137
Title: Biosensor design for infectious, water-borne agents
Investigators: Beitle, Robert
Institution: University of Arkansas - Fayetteville
EPA Project Officer: Page, Angela
Phase: I
Project Period: September 1, 2015 through August 31, 2016
Project Amount: $14,943
RFA: P3 Awards: A National Student Design Competition for Sustainability Focusing on People, Prosperity and the Planet (2015) RFA Text |  Recipients Lists
Research Category: P3 Awards , Pollution Prevention/Sustainable Development , Sustainability , P3 Challenge Area - Water

Objective:

The United Nations estimates that water-borne illness accounts for 80% of deaths in developing nations where nearly 1 in 6 do not have access to clean water [1]. Annually more than 200 million people in developed and developing countries are affected by floods, earthquakes, and other natural disasters that often interrupt water and electricity services for extended periods. As an example, consider the people of Louisiana and the impact of hurricane Katrina. During this emergency, at minimum 2.4 million people were without access to safe drinking water [2]. When testing water quality, “microbiological results typically require a minimum of 24 hours to complete”5 due to reliance on the traditional and inexpensive cultivation method. The World Health Organization describes LAMP as an “attractive diagnostic platform for resource-poor settings: it is fast (15-40 min), isothermal (requiring only a heat block), robust to inhibitors and reaction conditions that usually adversely affect PCR methods, and it generates a result that can be detected with the naked eye.” In 2011, Eiken Chemical Co. released the first LAMP-based tuberculosis detection kit designed to be simple enough and inexpensive enough to implement in resource-limited settings.1,15  Two important considerations provide motivation for our P3 effort:

 

  • Although LAMP reagent/assay kits have been developed for a number of infectious diseases, there has been limited focus on the development of LAMP-based diagnostic for water-borne pathogens, and
     
  • LAMP-based diagnostic platforms traditionally rely on conventional-PCR devices which are bulky and not easily deployed in a resource-poor environment.  A resource-poor environment is one that lacks access to electricity and lab equipment.

 

To wit, the overall objective of Phase I was to develop a low-cost, LAMP-based diagnostic platform capable of detecting the presence of water-borne pathogens in resource-poor environments by individuals with a modicum of formal training with the device.  The principal technical objectives of Phase I included:

Heating (1)

A method for providing heat to the device had to be designed and assessed.

Visualization (2)

A dye that could provide a positive result when the water-borne agent is present had to be selected.

LAMP (3)

A primer set for the model water-borne agent had to be designed for the ipaH gene shared in enteroinvasive E.coli and Shigella flexina.

While there were three technical objectives proposed in Phase I of the project, the overarching goal was to construct a prototype capable of demonstrating effective temperature control for demonstration of LAMP.

Summary/Accomplishments (Outputs/Outcomes):

Heating (1)

The objective of the device is to heat a sample of water containing LAMP reagents to 65 °C such that DNA replication can be initiated and sustained for up to 45 minutes. Initially, three heating methods were considered: chemical heating, disposable microbatteries, and a rechargeable battery pack, the last method being chosen for this proposal. The case of the device is 3D-printed and contains a phase-change material, a heating wire, and a rack containing the water vials.

The rechargeable battery pack was chosen as an appropriate heating method because it minimizes waste, increases sustainability, and most importantly, may be recharged easily.  In terms of waste / sustainability, while chemical heating methods like iron oxidation or forced crystallization are inexpensive and easily provide enough heat for a LAMP reaction, (i) the energy output cannot be precisely controlled, (ii) environmental factors like humidity and surrounding temperature cause these materials to fail, and most importantly (iii) require disposal when spent. Note that while microbatteries like the ones that are used to power GPS trackers in fish would provide improved control over power output, they do not provide nearly enough energy to sustain a LAMP reaction and still would require disposal of Cd or similar metal.

Our final heating device consisted of a battery pack that provides current through a nichrome heating wire via a USB cable. The USB connector provides attachment to the battery, and as will be described in our Phase II proposal, is integral to the plans we have for the next phase of the prototype.  At a diameter of 0.3 mm, nichrome has a resistivity of 15.4 Ω/m so that the resulting current through the heating element is 1.5 A for a wire length of 22 cm. Since USB devices operate at 5 V, the total power required of the device is a mere 7.5 W. 

Once the heating method was chosen and constructed, the next phase of the development consisted of designing a housing capable of containing not only the nichrome element, but also a material that when used in the prototype virtually eliminates the need for thermocouple-based temperature control.  As previously mentioned, virtually all useful LAMP-based detection methods still employ a PCR thermocycler.  These devices, while capable of precise temperature control and cycling, use advanced process control and electronics to maintain temperature.  In contrast, our prototype design uses a phase-change material to act as a thermal ballast.  Phase-change materials are wax-like substances that upon initial melting, release heat as they are cooled back to a solid state.  This solid / liquid transition provides excellent temperature control without the use of a thermocouple or ancillary electronics.  In other words, the inclusion of phase-change material in the design greatly drives simplicity, which in turn keeps cost down and eliminates the use of electronics which eventually may end up in a landfill.  

Several prototype devices were constructed via 3D printing and assessed for the following:

  • Reasonable start-time required to melt the phase-change material obtained from Entropy Solutions LLC.
  • Ability to monitor the melt transition visually.
  • Ability to maintain temperature at a value capable of supporting LAMP.

With regards to the phase-change material, the company provided us with a material capable of maintaining 63 °C, which is a reasonable temperature for LAMP.

To examine the heating characteristics, the device temperature was raised to approximately 65 °C.  Once the device reached this temperature, evident by the melting of the phase-change material, a 50-µL Eppendorf tubes containing a water sample and thermocouple was placed in the aluminum block. During the test phase, the biosensor’s temperature needs to be maintained between 63-67 °C, a reliable range wherein DNA replication can occur. Temperature control is achieved simply by turning the battery pack on and off, observing the condition of the Phase-change material.

Examination of the temperature profile indicated a degree of success with the first prototype, as a temperature of 62 °C +/- 2 °C could be easily obtained.  Figure 3 illustrates one simulated LAMP cycle for thirty minutes.  Other formats were examined but rejected because of long heat up times [ref].  The final functional prototype #1 (Figure 4) included the addition of a thermochromic material that changed from RED to CLEAR at 62 °C, a small round footprint, and metallized polyethylene terephthalate lining to help reduce heat loss.  This device required approximately 30 minutes of startup heating.

Our first functional prototype, “hockey puck”, was determined to lose heat to the surroundings via the aluminum that surrounded the tubes and the four windows that allowed for observation of the phase-change material.  A second functional prototype was printed that simply had holes for the microcentrifuge tubes, eliminating the aluminum block, and a single window .

The aluminum block was removed in prototype #2 and the PCR tubes were put directly into the phase change material. In order to evenly heat, the holes were alightd and the device was made taller.  This design required slightly more wire, with half of the wire as nichrome and the other half copper. The nichrome wire acted as a resistor and gave off heat, while the copper simply conducted the charge. This modification to the wire cut down on the energy required to heat the phase change material to its desired temperature: 63 °C. This change made a significant impact in the time it took to heat the phase change material during start up and PCR. Due to time constraints, a single termperature characterization was completed with prototype #2.  Figure 6 demonstrates that this simple design maintained temperature, 63 °C +/- 2 °C .

Visualization (2)

Fluorescent dyes are the most common method of indicating the presence of DNA post-PCR. The function of these dyes is to bind to the sugar-phosphate backbone of DNA: the chemical structure of the dye (a positively charged compound) adheres to the sugar-phosphate backbone of DNA (a negatively charged compound). When exposed to a certain wavelength of light – its excitation wavelength – the dye will emit a specific wavelength of light. Therefore, a sample containing DNA will exhibit a fluorescence which can be visually observed. Originally, the project aimed to use SYBR Green, a well-known and widely-used dye for DNA quantification purposes. However, a characteristic of the majority of fluorescent dyes – including SYBR Green – is their inhibition of the DNA replication process as a result of their bond to the chemical structure of DNA. In order to simplify the device and minimize steps in the LAMP process, a new dye – EvaGreen – was chosen that is specifically designed for PCR applications. This allowed the dye to be added to the solution pre-PCR with no inhibition of DNA replication.

There are several unique characteristics of EvaGreen dye that make it useful for LAMP applications. The key property of EvaGreen is that it is less inhibitory toward PCR and less likely to cause nonspecific amplification. As a result, EvaGreen can be used at a much higher concentration, resulting in a more robust signal and eliminating dye redistribution problems. The excitation and emission wavelengths of EvaGreen are comparable to commonly-used dyes: both are within the visible spectrum of light, making a sample containing DNA visible to the human eye. Another beneficial property of the dye is its safety for use and disposal. EvaGreen is non-mutagenic and non-cytotoxic; it is also completely cell-impermeable, a factor responsible for its low toxicity. These features are significant because typical DNA dyes like SYBR Green are powerful mutation enhancers, inhibiting the natural DNA repairing mechanism in cells. The toxicity of SYBR Green is associated with its ability to enter cells rapidly. Finally, EvaGreen is classified as nonhazardous for drain disposal, and is classified as nonhazardous to aquatic life. As a result, the selected dye is favorable for the intended use and provides secondary environmental advantages over standard fluorescent dyes.

To prove that EvaGreen is able to be visually observed, a standard was prepared with known quantities of DNA and dye. Concentrations of DNA ranged from 20uL to 0.1uL, and a constant 5uL of EvaGreen was added to every sample, totaling 25uL each sample. The standard shows that at relatively high concentrations of DNA, the visual signal is very strong and can be observed easily (Figure 7). At concentrations of DNA between 0.5uL and 0.1uL, the signal becomes visually unreadable. Thus, the target DNA range for PCR amplification is between 0.5uL and 1uL of DNA, which will make the sample easily observed.

LAMP (3)

Since the ipaH gene is common to enteroinvasive E.coli and S. flexina, it was chosen as a measure of water-borne pathogen contamination.  LAMP requires six DNA primers to amplify the DNA target.  Primer Explorer was used to design six primer sets for ipaH.

 

Conclusions:

Our Phase I project demonstrated that virtually all electronic components of a LAMP-based water borne pathogen detection system may be constructed based on the unique properties of phase-change materials.  The 3-D printed prototypes, envisioned to be close to a final design, cost less than $5.00 in material cost, making this system attractive as a means to provide this method of testing to both point-of-care and/or low resource settings.  If selected for Phase II, the group plans to test for ipaH in control (DNA only) and actual water samples, refine the prototype and develop a simple set of instructional material for its use in both classroom and real-world environments, and finally make the device available to healthcare professionals and those responsible for water quality assessment.