Grantee Research Project Results
Final Report: Aptamer Capture and Optical Interferometric Detection of Cyanobacteria
EPA Grant Number: R833839Title: Aptamer Capture and Optical Interferometric Detection of Cyanobacteria
Investigators: Campbell, Daniel P , Ellington, Andy , Xu, Jie
Institution: Georgia Institute of Technology , The University of Texas at Austin
EPA Project Officer: Aja, Hayley
Project Period: June 1, 2008 through April 30, 2011 (Extended to January 31, 2013)
Project Amount: $600,000
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens and Cyanobacteria and Their Toxins in Drinking Water (2007) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The objective of this program is to take the optical interferometric sensor that has been under development at the Georgia Tech Research Institute for the past 2 decades and configured it to detect and monitor a series of cyanobacterial toxins. After initial testing with known antibodies for a series of cyanobacterial toxins, the detection scheme will convert over to aptamers to provide a more rugged, reusable sensor for field deployment.
Cyanobacterial toxins have been recognized as a potential hazard in drinking water throughout the world. These bacteria are ubiquitous, growing wherever the conditions are favorable that includes almost any standing water one can imagine such as a freshwater pond, a swampy area, a dog’s water bowl and even one’s family’s toilet. Cyanobacterial toxins have been categorized into three groups based on their chemical structures. The toxins with highest priority are the neurotoxins anatoxin-a, cylindrospermopsin and most frequently found microcystin-LR. Their chemical structures of the more common toxins are shown in Figure 1.
The optical interferometric sensor provides a rapid, field-deployable and inexpensive device that can be adapted for detecting of cyanobacterial toxins. The GTRI interferometric optical sensor has been shown to be capable of detecting a wide variety of chemical and biological species. The sensor responds quickly with high sensitivity and specificity, and provides a direct measurement with no additional steps or consumable reagents. Some of the advantages of this sensor system for environmental applications are its low initial and per-sample cost, the ability to detect multiple analytes simultaneously, and the speed and sensitivity of detection. Systems can be designed for mobile, onsite field analysis with instant results or in situ monitoring with data logging and communication to a monitoring base.
In this project, the GTRI sensor will be first evaluated for detecting the cyanobacterial toxins using known antibodies as the recognition element that will bind with the toxins. In this detection scheme, the antibodies are covalently linked to the optical sensor’s surface. When the toxin is present it will bind to the recognition element causing a change in the refractive index. This refractive index change is detected by the waveguide interferometer and is converted after calibration to a concentration of the compound or species. Examples of this detection scheme employing this optical interferometer have included the detection of biotin with an avidin as the recognition element, and the detection of ricin a-chain, a protein toxin, employing an aptamer detection element, among countless other examples. After evaluating the sensor’s ability to detect the cyanobacterial toxins using known commercially available antibodies, the sensor’s recognition element will be changed over to nucleic acid-based aptamers as the recognition element. The aptamer provides a much more durable, reusable and reproducible recognition element than that of antibodies.
Summary/Accomplishments (Outputs/Outcomes):
Interferometric Sensor
Optical biosensors have been the subject of intense interest over the past 2 decades. This is due to the numerous advantages provided by optical methods, such as they can be miniaturized, have multiplexing capabilities, and because optical biosensor technology combines rapid response times with high sensitivity for analyte evaluation. One such emerging technology is the planar waveguide evanescent sensor combined with state-of-the-art surface functionalization chemistries. The waveguide evanescent sensor relies on monitoring the perturbation of the planar waveguide mode effective index resulting from the interaction of the evanescent tail of the mode with a changing ambient medium. For many applications, this medium is a solution containing molecules such as proteins, DNA, RNA, antigens or antibodies, which bind to a layer of receptor molecules immobilized on the waveguide surface. Several transducers have been developed using evanescent field for sensing including interferometer,1 grating coupler2 and SPR.3 The interferometric sensors generally have the highest sensitivity, compared to SPR and grating coupler-based sensors. This is mainly based on possibility of using large interaction length values resulting in enhanced sensitivity. A typical value of 1.5 cm is used for the interferometer, being an order of magnitude larger than the typical values of 1 mm for the grating coupler and 10-100 μm for the SPR sensors. We have developed a planar waveguide interferometric sensor capable of simultaneously detecting a wide array of analytes, including toxins, viruses, spores and whole cell bacteria as well as chemical threats in a fast, direct and sensitive method.1, 4 The waveguide is fabricated with an array of sensing interferometers capable of detecting multiple analytes at once and requiring only a single light source and detector to analyze the multiple sampling channels fabricated onto a single 1.6 x 3.3 cm glass substrate. Sensitivities for this sensor are in the pg/ml, <1000 cfu/ml, <.0005 HA units/ml and/or ppt levels of proteins, spores/cells, viruses and/or chemicals, respectively.
At the heart of the GTRI optical sensor is a planar optical waveguide. Planar waveguides are related to fiber optic waveguides in that a high refractive index material is used to guide the light within its confines through total internal reflection. Whereas a fiber optic’s high refractive index core is surrounded by a lower refractive index cladding, the planar waveguide has the high refractive index waveguiding layer deposited on top of a lower refractive index planar substrate. Planar waveguides have evanescent fields, the tail of the electromagnetic field associated with the propagation of light, sensitive to index of refraction changes in the volume immediately above the waveguide surface. These fields extend up to 500 nanometers (nm) above the surface. Coupling a bioreceptor, such as an antibody, to the waveguide surface provides the basis for a biosensor (Figure 1). Briefly, light from a diode laser is coupled into the planar waveguide with a grating etched in the glass substrate. When a test solution containing the target antigen is introduced to the functionalized waveguide, the antigen binds to the receptor and displaces water near the waveguide surface causing a change in the velocity of the propagating light. To measure this change, an adjacent but unperturbed reference beam is optically combined with the sensing beam (Mach-Zender interferometer), creating an interference pattern of alternating dark and light fringes (Figure 2). When refractive index changes occur in the sensing arm, the interference pattern shifts, producing a resultant change in the relative phase that is measured using a Fourier transform algorithm indicating the presence and concentration of the target antigen. No secondary or reporter antibody is required. With detection sensitivities on the order of 0.01 radians, refractive index changes of less than 10-6 can be measured. This design provides a direct, label-free and reagentless means of detection.
In the current prototype, off-the-shelf components, including a laser diode light source, a monochrome CCD camera and a flow cell, are integrated in a 30 x 12 x 12 cm3 portable case (Figure 3). External components consist of a sample delivery device (peristaltic pump) and data collection computer. Power is supplied through a Firewire connection.
Waveguide Fabrication
Single-interferometer waveguides have to be redesigned and fabricated for toxin detection and validation due to the more compact design of the second-generation prototype. Previous biosensors can’t be used in this newer prototype because the compact optical design decreases the fringe spacing of the interference pattern to 20 μm (it can be calculated lf/d, where l is the wavelength, f is the focusing length and d is the separation of the two output gratings) if an old biochip (d is 1 mm) was used. Only 4 pixels (with a pixel size of 5 μm) on the CCD camera were used for one fringe, which makes light intensity tracking less accurate. In order to produce an increased fringe spacing to cover enough pixels for accurate, real-time light intensity tracking, a new single-interferometer waveguide has been designed and a grating mask has been fabricated. The design is shown in Figure 4. The distance between sampling and reference channel is decreased to 0.3 mm. By doing this, the fringe spacing is increased to 67 μm so 13 pixels can be covered for real-time intensity tracking.
The substrate for the optical chip is made of fused quartz. The output/input grating couplers are produced by inductively coupled plasma (ICP) etching a 0.720 mm period grating onto the substrate surface to a depth equivalent to the grating period. A silicon nitride waveguide layer (thickness = 0.140 mm) is then deposited using plasma-enhanced chemical vapor deposition (PECVD). This is then covered with a thin film (200 Å) of silicon dioxide to protect the waveguide and allow attachment of chemically selective films. Finally, the two arms of the interferometer are defined by depositing a thick (5,000 Å) silicon dioxide layer via ion-assisted deposition (IAD) everywhere except the sensing regions. The planar optical waveguide platform allows for multiple, parallel interferometers on the 1.6 x 3.3 cm glass substrate. A different sensing film deposited on each channel can provide for multiple analyte sensing, interferant cancellation, patterned outputs for analyte identification or extended dynamic range.
Sensitivity calibration: One arm of the interferometer was coated with a polymer film with thickness greater than 500 nm. Refractive indexes of salt water (NaCl) and deionized water were measured using a refractometer. Then, deionized water was flowed over the waveguide surface using a peristaltic pump to register a flat baseline before salt water was introduced. The phase change caused by the change of refractive index from deionized water to salt water was measured and used for the sensitivity measurement based on the equation:
where is the measured phase change, l is the length of the channel, which is 1.75 cm, and is the refractive index changes between salt water and deionized water. Measured biochip sensitivities were listed in Table 1. Sensitivity variation for the biochips is ~ 10%.
Table 1. Waveguide Sensitivity Calibration
Waveguide |
Average of measured Sensitivity |
Standard deviation |
P109 |
3.71 |
0.13 |
P110 |
4.40 |
0.14 |
P113 |
4.10 |
0.03 |
P116 |
3.96 |
0.32 |
P117 |
4.33 |
0.17 |
P118 |
4.34 |
0.09 |
P121 |
4.36 |
0.03 |
Bioreceptor Immobilization
The waveguides were cleaned with a 2% Micro-90 solution (International Products Corporation, Burlington, NJ) and rinsed with DI water, followed by immersion in chromic acid for 30 minutes at 70-80°C, rinsing with DI water and drying with a stream of clean air. The waveguides were incubated with 2% MTS (3-mercaptotrimethoxysilane) solution in anhydrous toluene for 1 hour under an argon atmosphere and then rinsed with toluene and dried. The silanized chips were then incubated with 2 mM MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester, prepared by dissolving 1 mg of MBS in 0.3 mL anhydrous DMSO then diluting to 1.5 mL with anhydrous ethanol) for 1 hour and rinsed with DI water. Next, 2 µL of monoclonal anti-microcystin IgG antibody (1 mg/mL, obtained from Enzo Life Science) and 2 µL of goat IgG (1 mg/mL) were applied to the sensing and reference channels respectively and allowed to sit for 1 hour in a humid chamber. Finally the waveguides were rinsed with DI water and stored in 1 M ethanolamine blocking buffer at 4°C until use. The immobilization chemistry was shown in Figure 5. The same immobilization chemistry was also used for amino-modified aptamers.
Microcystin-LR Detection Using Antibody-Coated Chips
Immediately prior to using, the prepared waveguides were removed from blocking buffer and rinsed with PBS. Analyte solutions were delivered to a flow cell using a peristaltic pump in a closed loop setup, shown in Figure 6. A running buffer consisting of PBS containing 0.1% TX-100 (PBS+) was flowed over the waveguide surface at 2 ml/min until a stable baseline was established. When drift was found to be less than 0.01 radians/min, the waveguides were ready for application of samples.
Microcystin-LR (standards were obtained from Envirologix) sample dilutions of 10 mL in PBS+ were introduced into the system and circulated in a closed loop over the biochip surface for about 10 minutes or until measurable signal was observed. Between samples, the waveguide was flushed with PBS+ to remove any nonspecifically bound species. Figure 7 shows the raw sensor responses to microcystin-LR in PBS+ buffer. Samples with 100 pg/mL rabbit IgG can be detected. The sensing assay is extremely sensitive. A concentration of 3 ppt microcystin-LR can be easily detected. In addition, the binding is reversible so the coated chip can be reused for many times without losing the detection sensitivity.
A linear calibration curve was obtained with the sensing responses to the concentration of the microcystin solution in the log scale, as shown in Figure 8. With the noise level of 0.01, a detection limit of 0.14 ppt is obtained, which is the most sensitive detection method compared to the literature-reported values. Table 2 summarizes the current methods and corresponding detection limits for microcystin.
- Table 2. Comparison of Methods Currently Available for Analysis of Microcystin Variants5
Methods |
Comments |
Limit of detection |
References |
Whole cell bioassay |
Measure LC50 value, qualitative, poor sensitivity and precision, ethical issues |
25 - 150 μg/kg (mouse) |
|
TLC |
Requires standard, low sensitivity, very easy |
0.01 ug |
|
ELISA |
Colorimetric, sensitive cross reaction, expensive |
PAb:2.5 μg/L, MAb:0.1 μg/L |
|
Protein phosphatase inhibition (PPI) assay |
Colorimetric, fluorometric, very sensitive, false positives, expensive |
Colorimetric: 0.25 -2.5 μg/L, Fluorometric: 0.1 μg/L |
|
HPLC |
Measure UV absorbance, ISO standard method, detection of individual variants, required standards, expensive, required trained personnel |
PDA/UV array: 100 μg/L, MS: 0.1 μg/L |
|
MALDI-TOF |
Identification of variants, structural elucidation, matrix interferences |
1 μg/L |
|
NEEIA |
Rapid, sensitive, disposable, portable, useful for site monitoring |
0.1 μg/L |
|
Chl-a fluorescence-based bioassay |
Rapid, sensitive, useful for different variants of MC |
0.01 μg/L |
|
Cl-immunoassay |
Wide linear range, highly sensitive, suitable for natural sample |
0.02 μg/L |
|
LC-MS |
Mass spectrometry, simultaneous separation and identification of variants, sensitive and specific, very expensive |
~ 0.02 μg/L |
|
LC-MS/MS |
Identification of variants, sensitive and specific, expensive |
0.0026 μg/L |
|
Gas chromatography (GC and GC/MS) |
Fluorescence detection, low sensitivity, tedious process |
0.0043 μg/L |
|
TRFIA |
Broad working range, highly sensitive and efficient |
5 x 10-6 μg/L |
|
Capillary electrophoresis (CE and CE-MS) |
Laser-induced fluorescence detection, low sensitivity needs further improvement |
|
|
NMR |
Structural determination of cyanotoxins, requires large amount of sample, requires pure sample, expensive |
|
|
Cantilever sensor |
Less stable |
1 ng/L |
|
Our work |
Fast, reusable, low cost |
140 pg/L |
|
Aptamer Specific to Microcystin
Protein antibodies are typically used as recognition elements for immunoassay-based cyanobacterial toxin detection and measurement. However, cyanobacterial toxins such as anatoxin are too small to invoke an immune response in animals on their own. In order to develop an antibody for a typical immunoassay one has to conjugate the toxin to a higher molecular weight protein. As a result, cross-reactivities have been reported. In addition, antibodies are inherently unstable in outdoor urban monitoring applications. As a cost-effective and easily engineered alternative, we will utilize aptamers, whenever possible, as recognition elements. Aptamers provide a durable, reusable, highly specific recognition element that can be readily attached to the waveguide surface.
The typical procedure for aptamer selection was shown in Figure 8. In short, a random sequence DNA pool is generated by chemical synthesis. The synthetic oligonucleotide typically contains a random core of 30 to 90 randomized residues, flanked by constant sequences that assist in enzymatic amplification, including in vitro transcription to generate an RNA pool. A random sequence DNA or RNA pool can be incubated with any target protein and those few sequences in the large population that bind the protein can be isolated by methods such as filtration or a chromatographic technique. The isolated nucleic acid-binding species are then amplified and used in additional rounds of selection and amplification. Multiple cycles of selection and amplification tend to winnow a pool of upwards of 1015 molecules to only those few species that have the highest affinities and specificities for a target protein. The Ellington lab has extensive experience in the selection of aptamers against targets ranging from small organics to peptides to proteins to supramolecular structures such as cells.30
Microcystin-specific aptamers with great binding affinity and specificity have been published recently.31 Two aptamers were chosen for our study due to their binding affinity and specificity. Their sequences and binding affinity were listed in Table 3. The aptamers were modified with amino functional group at the 5’ end and synthesized by TriLink Biotechnologies (San Diego, CA).
Table 3. ssDNA Sequences Selected for Microcystin Detection
Name |
Sequence |
Kd (nM) |
RC4 |
CACG CACA GAAG ACAC CTAC AGGG CCAG ATCA CAAT CGGT TAGT GAAC TCGT ACGG CGCG |
76±13 |
RC6 |
CACG CAAC AACA CAAC ATGC CCAG CGCC TGGA ACAT ATCC TATG AGTT AGTC CGCC CACA |
61±4 |
Aptamer-Based Microcystin Detection
Because of the presence of amino function group, the aptamer molecules can be immobilized on the waveguide surface using the same conjugation chemistry as shown in Figure 5. Briefly, 1 μmol of aptamer RC4 and RC6 were dissolved in 1 mL of PBS. Two μL of each prepared aptamer solutions were applied on top of the waveguide and incubated for 1 hour for aptamer immobilization. Then, the waveguides were rinsed and stored in 1 M ethanolamine blocking buffer at 4°C until use.
PBS+ was used to register baseline. When the drift is smaller than 0.1 radians in 10 minutes, the sample was switched and flowed over the waveguide surface for sensing measurement. Typical sensing responses for microcystin using a RC4- and RC6-coated waveguide were shown in Figure 9 and 10, respectively. Although a sensitive response can be obtained for 333 ppt of toxin and response is reversible, the response is not correlated with the concentration of the toxin. Individual concentrations were extracted out from Figure 10 and plotted in Figure 11. It can be seen clearly that the responses were not very dependent on the concentration. More investigation are underway to examine the issues with the aptamers.
Conclusions:
A sensitive method has been developed for microcystin detection using the interferometric based optical sensor. A detection limit of 140 pg/L has been obtained with a monoclonal antibody-coated waveguide. The response is fast and reversible so the sensing waveguide can be reused many times. In addition, the sensor prototype is compact and field-usable so the microcystin monitoring in the field can be achieved. Aptamers specific to microcystin were synthesized by Trilink Biotechnology and coated on the waveguide. The measurements for microcystin were explored but failed. The issues associated with aptamers are still under investigation.
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Supplemental Keywords:
Interferometric sensor, aptamer, microcystin, waveguide, water, environmental chemistry, health risk assessment, environmental monitoring, drinking water, environmental engineering, microbial contamination, monitoring, real time analysis, microbial risk assessment, gene microarray assay, aquatic organisms, other - risk assessment, early warning, drinking water contaminants, cyanobacteria;, RFA, Scientific Discipline, INTERNATIONAL COOPERATION, Water, Environmental Chemistry, Health Risk Assessment, Environmental Monitoring, Environmental Engineering, Drinking Water, microbial contamination, microbial risk assessment, monitoring, real time analysis, gene microarray assay, aquatic organisms, other - risk assessment, early warning, drinking water contaminants, drinking water systemProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.