Aptasensors: Beyond The Aptamer Molecule
Our Technology relies on the application of the recognition potential exhibited by aptamers in a sensing device. This page contents all the concepts involving our biosensing technologies. The hardware that embraces our aptamers and make them able to generate a signal after the recognition of their target molecules.
In order to create the best possible solution to the social problem that cholera is, we have used all the hints provided by our Human practices research. Eventually we have concluded that our technology must consist on two different architectures: An electrochemical sensor and a paper-strip based sensor.
We have built a mini potentiostat device, capable of executing some of the most common electrochemical analysis techniques. Our device is focused on performing voltamperometric analysis, such as cyclic voltammetry or linear sweep.
Why an electrochemical sensor?
An electrochemical sensor allows our technology going further. Electrochemical techniques allow for a high sensibility and accuracy, allowing quantitative detection. What’s more, using a microelectronic-based device, gives our sensor the potential to be easily transported and obtaining real-time sensing readings. That’s an enormous advantage compared with the hours to days consumed with the traditional identification methods.
We aim to measure the amount of a pathogen present in an aqueous sample, starting with Vibrio cholerae as first step. The measure will be performed via Square Wave Voltammetry (SWV) analysis, employing an aptamer-modified microelectrode. Using an expendable standardized microelectrode, it’s the perfect way to measure as many different targets as aptamer-modified electrodes you have, by just replacing the electrode. That gives our device the opportunity to be used as an almost universal tool for aptamer-based-sensing, granting our technology the potential to be scalable on a continuous way.
Square Wave Voltammetry
As most of the voltammetry techniques, SWV relies on the application of a constant potential during short intervals of time, and increasing the potential periodically, alternating periods of “low” and “high” potential. The current passing trough the sample via the working electrode is measured at the end of each constant potential steps. The oxidation/reduction of a redox active specie in the solution, can be measured as the difference between the current measured during “low potential” and “high potential” periods. The “differential current peak” can be correlated with the concentration of the species.
Immobilizing an aptamer on the electrode surface, leads to a reduction in the measured current. When the immobilized aptamer changes its conformation in presence of the targeted molecule, the measured current it’s also altered. That fact allows to perform quantitative measurements, correlating the measured differential current trough the electrode with the analyte present in the sample for a constant concentration of redox active species. [4]
Our Device
Building our own potentiostat based on the open-source project CheapStat [1], have allowed us to optimize the cost and size of the final device. The final version cost less than 40 $, and the size have been divided by a twofold factor. [2],[3].
First prototype of out potentiostat, with a microelectrode.
Our potentiostat aims to be a powerful and versatile sensing tool. Due to it’s reduced cost and size, its potential applications can vary from accurate diagnosis in little medical facilities to big industry usage in real time quality controls.
Final version of out potentiostat, mounted in methacrylate.
We have built a mini potentiostat device, capable of executing some of the most common electrochemical-analysis techniques. Our device is focused on performing voltamperometric analysis, such as cyclic voltammetry or linear sweep.
Experiments and Protocols
Details about the potentiostat development process, blueprints and comments about it can be found in the team’s GitHub. Also the programmed code for performing square wave voltammetry readings and other techniques is available and detailed in our GitHub.
Experimental Results and discussion
After buildup and programming of our potentiostat, we have tested that our system works properly. Since past year we probed that aptamer modified electrodes are capable of generating a significative analytical signal, we have focused on proving that our potentiostat works properly.
We have tried the potentiostat with water and different levels of a redox active species such as ferricyanide, the direct reading results of our program can be visualized as an Intensity – potential graph. Figures 1-4 depicts all of that information.
Figure 1.
Raw potentiostat results using distilled water.
Figure 2.
Raw potentiostat results at 0.25 mM ferricyanide.
Figure 3
. Raw potentiostat results at 0.5 mM ferricyanide.
Figure 4.
Square Wave Results for a test assay using ferricyanide as redox active species.
After analyzing the results of the different measurement experiments, we can assert that there is an obvious difference in signal between the negative controls (deionized water) and the positive samples. The typical current peak signal is just observed when the analyte is present. Likewise, after processing the data considering the corrected current peak intensity, we have found a direct correlation with concentration of analyte and the peak instensity signal.
Future Steps
We have created a potentiostat based sensor and we have proved that the system works properly for performing electroanalytical detections. Basing on the work developed during the past year in the IoBT iGem project, we can conclude that it is possible to attach an aptamer to a microelectrode, allowing for quantitative detection of the targeted molecule.
However, that’s just the beginning of an exciting new technology which aims to serve as a standardized tool for scalable biological sensing. Future iGemer’s or any person interested in following our development procedure will be able to take all the technology we have develop and continue our effort. Next logical steps will be the establishment of a standarized collection of different aptamer-modified electrodes and testing the analytical correlations and parameters which will allow the building of functioning sensing platform.
Conclusions and Further Improvement
Summarizing all the knowledge acquired, we can conclude that aptamers are molecules with an enormous potential able to be applied in many different sensing technological approaches.
Potentiostat
We have been developed a fully working electrochemical device, with expendable aptamer-modified microelectrodes. That system can be easily used and reproduced according the blueprints and instructions available in our GitHub. Due to it’s reduced cost it will become an useful instrument which will save a lot of time, resources and even human lives.
References
[1] Rowe, A., Bonham, A., White, R., Zimmer, M., Yadgar, R., Hobza, T., Honea, J., Ben-Yaacov, I. ad Plaxco, K. (2011). “CheapStat: An Open-Source, “Do-It-Yourself” Potentiostat for Analytical and Educational Applications”. PLoS ONE, [online] 6(9), p.e23783.
[2] Ainla, A., Mousavi, M., Tsaloglou, M., Redston, J., Bell, J., Fernández-Abedul, M. and Whitesides, G. (2018). “Open-Source Potentiostat for Wireless Electrochemical Detection with Smartphones”. Analytical Chemistry, [online] 90(10), pp.6240-6246.
[3] M. Dryden and A. Wheeler, "DStat: A Versatile, Open-Source Potentiostat for Electroanalysis and Integration", PLOS ONE, vol. 10, no. 10, p. e0140349, 2015.
[4] L. Li, H. Zhao, Z. Chen, X. Mu, and L. Guo, “Aptamer biosensor for label-free square-wave voltammetry detection of angiogenin,” Biosens Bioelectron, vol. 30, no. 1, pp. 261–266, Dec. 2011.
[5] S. Zhao, S. Wang, S. Zhang, J. Liu, and Y. Dong, “State of the art: Lateral flow assay (LFA) biosensor for on-site rapid detection,” Chinese Chemical Letters, vol. 29, no. 11, pp. 1567–1577, Nov. 2018.
[6] C. Carrell et al., “Beyond the lateral flow assay: A review of paper-based microfluidics,” Microelectronic Engineering, vol. 206, pp. 45–54, Feb. 2019.
[7] M. Jauset-Rubio, M. S. El-Shahawi, A. S. Bashammakh, A. O. Alyoubi, and C. K. O’Sullivan, “Development of Aptamer-Based Lateral Flow Assay Methods,” in Aptamers for Analytical Applications, Y. Dong, Ed. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2018, pp. 273–299.
[8]. G. Bruno, “Application of DNA Aptamers and Quantum Dots to Lateral Flow Test Strips for Detection of Foodborne Pathogens with Improved Sensitivity versus Colloidal Gold,” Pathogens, vol. 3, no. 2, pp. 341–355, Apr. 2014.
[9] E. Frohnmeyer et al., “Aptamer lateral flow assays for rapid and sensitive detection of cholera toxin,” Analyst, vol. 144, no. 5, pp. 1840–1849, 2019.
[10] F. Cimaglia et al., “Detection of mycobacterial DNA by a specific and simple lateral flow assay incorporating cadmium selenide quantum dots,” Molecular and Cellular Probes, vol. 29, no. 6, pp. 534–536, Dec. 2015.
[11] X. Mao, W. Wang, and T. E. Du, “Dry-reagent nucleic acid biosensor based on blue dye doped latex beads and lateral flow strip,” Talanta, vol. 114, pp. 248–253, Sep. 2013.
[12] G. Liu, A. Gurung, and W. Qiu, “Lateral Flow Aptasensor for Simultaneous Detection of Platelet-Derived Growth Factor-BB (PDGF-BB) and Thrombin,” Molecules, vol. 24, no. 4, p. 756, Feb. 2019.
[13] P. Porschewski, M. A.-M. Grättinger, K. Klenzke, A. Erpenbach, M. R. Blind, and F. Schäfer, “Using Aptamers as Capture Reagents in Bead-Based Assay Systems for Diagnostics and Hit Identification,” J Biomol Screen, vol. 11, no. 7, pp. 773–781, Oct. 2006.
[14] S. Marton, F. Cleto, M. A. Krieger, and J. Cardoso, “Isolation of an Aptamer that Binds Specifically to E. coli,” PLoS ONE, vol. 11, no. 4, p. e0153637, Apr. 2016.
[15] I. M. Ferreira, C. M. de Souza Lacerda, L. S. de Faria, C. R. Corrêa, and A. S. R. de Andrade, “Selection of Peptidoglycan-Specific Aptamers for Bacterial Cells Identification,” Appl Biochem Biotechnol, vol. 174, no. 7, pp. 2548–2556, Dec. 2014.
[16] L. Wang et al., “An aptamer-based chromatographic strip assay for sensitive toxin semi-quantitative detection,” Biosensors and Bioelectronics, vol. 26, no. 6, pp. 3059–3062, Feb. 2011.
[17] C. A. Holstein et al., “Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers,” Anal Bioanal Chem, vol. 408, no. 5, pp. 1335–1346, Feb. 2016.
[18] M. A. Mansfield, “Estapor® Microspheres in Lateral Flow Assays,” p. 27. [19] G. T. Hermanson, “Nucleic Acid and Oligonucleotide Modification and Conjugation,” in Bioconjugate Techniques, Elsevier, 2008, pp. 969–1002.
[20] J. Orlans et al., “iGEM REPORT: Gotta Detect ‘Em All: a multi-STI sensor based on aptamers,” Plos blogs, May 2017.
[21] K. E. Boehle et al., “Utilizing Paper-Based Devices for Antimicrobial-Resistant Bacteria Detection,” Angew. Chem. Int. Ed., vol. 56, no. 24, pp. 6886–6890, Jun. 2017.
[2] Ainla, A., Mousavi, M., Tsaloglou, M., Redston, J., Bell, J., Fernández-Abedul, M. and Whitesides, G. (2018). “Open-Source Potentiostat for Wireless Electrochemical Detection with Smartphones”. Analytical Chemistry, [online] 90(10), pp.6240-6246.
[3] M. Dryden and A. Wheeler, "DStat: A Versatile, Open-Source Potentiostat for Electroanalysis and Integration", PLOS ONE, vol. 10, no. 10, p. e0140349, 2015.
[4] L. Li, H. Zhao, Z. Chen, X. Mu, and L. Guo, “Aptamer biosensor for label-free square-wave voltammetry detection of angiogenin,” Biosens Bioelectron, vol. 30, no. 1, pp. 261–266, Dec. 2011.
[5] S. Zhao, S. Wang, S. Zhang, J. Liu, and Y. Dong, “State of the art: Lateral flow assay (LFA) biosensor for on-site rapid detection,” Chinese Chemical Letters, vol. 29, no. 11, pp. 1567–1577, Nov. 2018.
[6] C. Carrell et al., “Beyond the lateral flow assay: A review of paper-based microfluidics,” Microelectronic Engineering, vol. 206, pp. 45–54, Feb. 2019.
[7] M. Jauset-Rubio, M. S. El-Shahawi, A. S. Bashammakh, A. O. Alyoubi, and C. K. O’Sullivan, “Development of Aptamer-Based Lateral Flow Assay Methods,” in Aptamers for Analytical Applications, Y. Dong, Ed. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2018, pp. 273–299.
[8]. G. Bruno, “Application of DNA Aptamers and Quantum Dots to Lateral Flow Test Strips for Detection of Foodborne Pathogens with Improved Sensitivity versus Colloidal Gold,” Pathogens, vol. 3, no. 2, pp. 341–355, Apr. 2014.
[9] E. Frohnmeyer et al., “Aptamer lateral flow assays for rapid and sensitive detection of cholera toxin,” Analyst, vol. 144, no. 5, pp. 1840–1849, 2019.
[10] F. Cimaglia et al., “Detection of mycobacterial DNA by a specific and simple lateral flow assay incorporating cadmium selenide quantum dots,” Molecular and Cellular Probes, vol. 29, no. 6, pp. 534–536, Dec. 2015.
[11] X. Mao, W. Wang, and T. E. Du, “Dry-reagent nucleic acid biosensor based on blue dye doped latex beads and lateral flow strip,” Talanta, vol. 114, pp. 248–253, Sep. 2013.
[12] G. Liu, A. Gurung, and W. Qiu, “Lateral Flow Aptasensor for Simultaneous Detection of Platelet-Derived Growth Factor-BB (PDGF-BB) and Thrombin,” Molecules, vol. 24, no. 4, p. 756, Feb. 2019.
[13] P. Porschewski, M. A.-M. Grättinger, K. Klenzke, A. Erpenbach, M. R. Blind, and F. Schäfer, “Using Aptamers as Capture Reagents in Bead-Based Assay Systems for Diagnostics and Hit Identification,” J Biomol Screen, vol. 11, no. 7, pp. 773–781, Oct. 2006.
[14] S. Marton, F. Cleto, M. A. Krieger, and J. Cardoso, “Isolation of an Aptamer that Binds Specifically to E. coli,” PLoS ONE, vol. 11, no. 4, p. e0153637, Apr. 2016.
[15] I. M. Ferreira, C. M. de Souza Lacerda, L. S. de Faria, C. R. Corrêa, and A. S. R. de Andrade, “Selection of Peptidoglycan-Specific Aptamers for Bacterial Cells Identification,” Appl Biochem Biotechnol, vol. 174, no. 7, pp. 2548–2556, Dec. 2014.
[16] L. Wang et al., “An aptamer-based chromatographic strip assay for sensitive toxin semi-quantitative detection,” Biosensors and Bioelectronics, vol. 26, no. 6, pp. 3059–3062, Feb. 2011.
[17] C. A. Holstein et al., “Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers,” Anal Bioanal Chem, vol. 408, no. 5, pp. 1335–1346, Feb. 2016.
[18] M. A. Mansfield, “Estapor® Microspheres in Lateral Flow Assays,” p. 27. [19] G. T. Hermanson, “Nucleic Acid and Oligonucleotide Modification and Conjugation,” in Bioconjugate Techniques, Elsevier, 2008, pp. 969–1002.
[20] J. Orlans et al., “iGEM REPORT: Gotta Detect ‘Em All: a multi-STI sensor based on aptamers,” Plos blogs, May 2017.
[21] K. E. Boehle et al., “Utilizing Paper-Based Devices for Antimicrobial-Resistant Bacteria Detection,” Angew. Chem. Int. Ed., vol. 56, no. 24, pp. 6886–6890, Jun. 2017.
According to the conclusions of our human practice research, we have designed a paper based sensor, adapting the widely studied format of lateral flow immunochromatography to aptamers.
Briefly, we have laid the groundwork, for scalable aptamer-based lateral flow rapid test development. Our objective is to build a lateral flow sensor for whole Vibrio Cholerae cells as starting point.
Why a paper-based sensor?
The Main advantage of using a paper-based sensor are its ease of use, cheap manufacturing cost, and the possibility of performing a test without any other infrastructure. In addition, working with that format, allows the easy development of the technology once optimized. Even with little resources will be possible to build the sensor.
Point of Care devices are nowadays an emerging technology which points to offer closer resolution of the user’s needs. We want to create a simple, handy and reliable sensing solution, which almost anyone without specialized knowledge can use. That’s the reason of why a Lateral Flow Paper Strip is the ideal format for accomplishing our purposes. [5],[6]
Sandwich Format Lateral Flow Assays
Typical lateral flow assays depend on antibodies as recognition molecules, despite its versatility, antibodies has some disadvantages that make that kind of devices lose efectiveness under certain conditions. Replacing antibodies with aptamers can greatly improve the performance of that assays, thanks to their properties. [7],[8],[9],[10],[11],[12],[13]
Aptamers are more stable under aggressive conditions such as high temperatures, humidity or long storage periods. Also have been proved that aptamers can offer greater sensitivity compared to antibodies. In addition, since it’s molecular nature, working with nucleic acids it’s much easier than antibodies, it implies a cheaper and simpler manipulation and production process.
“Sandwich format” is one of the most used general configurations for lateral flow assays. It involves two different sensing molecules: the capture and the detection ones. That detection system works thanks to multiple steps which happens sequentially while the sample runs trough the membrane.
Targeted Bacteria
Latex Beads
Capture Aptamer
Streptavidin
Test aptamer
Control DNA Probe (Poly-T)
Overview of a sandwich format lateral flow assay.
Design of our device
To build our aptamer lateral flow device, we have divided the process in the main different parts it is composed and performing experimentation with each one of them separately: Aptamers Immobilization to the membrane, conjugation of the capture aptamer to reporter particles and optimization of the pads and experimental conditions has been the followed sequence.
Experiments and Protocols
In the following section we have collected all the protocols followed for the performance of each experimental step, involving our aptamer-based lateral flow development procedure.
Experimental Results and discussion
Microfluidic Implementation
The wax1 protocol describes a wax-printing method for microfluidic channels creation on a analytical membrane. Relying on 3d-printed stamps and thermal permeabilization. The procedure demonstrated to work, but with a poor reproducibility. Because of that we have built a new Opentrons-2 wax module and adapted our wax stamp system to fully automatize the priming step. Figures 5 and 6 shows the wax-priming procedure and the sealed membranes.
Figure 5.
Manually wax-printed membranes. On the left it’s appreciable channel narrowing because of low reproducibility of the printing
Figure 6.
3D Design of a stamp for membrane wax priming.
The developed wax-priming protocol has been adapted for using with different stamp designs, which have been a useful tool for performing many of our experiments. All hardware, 3d part designs and manufacturing details can be found in the team’s GitHub.
Reagents Immobilization
Firstly, we have adapted the ELONA assay for testing the designed streptavidin-biotin tagged DNA, immobilization system. This experiments aims to elucidate if it’s possible to tightly attach our DNA probes to the membrane. We have also demonstrated that complementary DNA interaction can develop within the membrane conditions. [17]
The Immob1 protocol results are shown in figure 7, Immob2 protocol results are depicted in figure 8.
Figure 7.
qualitative ELONA results, employing a 0.45 µm nitrocellulose membrane. a) Immobilized streptavidin and interaction with biotinDNA-RC-Dig conjugatd. b) Immobilized streptavidin followed by addition of biotinDNA, drying and further RC-Dig deposition. c) Immobilized streptavidin-biotinDNA conjugate, followed by addition of RC-Dig. d) Only streptavidin, e) streptavidin, BSA block and antibody, f) streptavidin and immobilized antibody positive control.
Figure 8.
Quantitative ELONA results. I) Indicates the nitrocellulose membrane used. Different amounts of streptavidin have been immobilized. Controls C1 (only streptavidin), C2 (streptavidin, BSA block and antibody), C3 (streptavidin and immobilized antibody), C4 (no antibody).
The intense color due to ABTS oxidation by the peroxidase conjugated with Anti-Digoxigenin antibody, let us conclude that effectively streptavidin can bind effectively to nitrocellulose membranes.
Attending to Figure 7 results, we have also determined that complementary DNA sequences can interact and get retained in the membrane. According with that results we have established the easiest procedure for DNA immobilization in the membrane, which is collected in the LFA1 (Lateral Flow Buildup) protocol.
Results depicted in figure 8 have allowed us to study the influence of membrane in streptavidin immobilization, and the sensitivity of the paper-ELONA assay. Figure 9 shows the results analysis, determining that streptavidin immobilization is dependent of the membrane pore size. Limit of detection for streptavidin detection have been established in 1 ug.
Figure 5.
Quantitative ELONA results. Smaller pore sizes offer a better retention of streptavidin within the membrane.
Latex beads aggregation management
Once tested the immobilization system, next step was learning how to manage nanoparticle behavior. Beads1 protocol details how different conditions affect the latex beads migration trough the analytical membrane.
Since our carboxylate modified latex beads are insoluble nanoparticles, stabilized in aqueous suspension helped by their charged surface groups, changing pH of the media can interfere with the particle stability. Other compounds such as metal ions can also affect beads promoting aggregation.
Figure 10 details how different buffers affect the migration of beads trough the membrane.
Figure 10.
Beads migration trough membrane. a) – d) 1% suspension in dH2O, PBS-T 0.05%, NaHCO3 100 mM pH 9.6 and MES 50 mM pH 5.9 respectively. e) – h) Migration after resuspending each of the previously mentioned bead suspensions in dH2O.
With this assay we have tested how changing the buffer in which beads are suspended can affect the particles dispersibility. When particles surface properties are altered, interaction with water can decrease, driving to interaction via Van der Waals forces between hydrophobic latex particles, eventually it can lead to irreversible aggregation between particles.
Short periods on conditions which favor latex aggregation doesn’t affect significatively the posterior behavior once the conditions change, however as it possible to observe in Figure 10 g), the buffer can also alter permanently the behavior of beads, even after resuspension. That’s the reason why guaranteeing a good beads dispersion and minimize the amount of time beads spent on aggressive conditions during conjugation it’s a key aspect to consider.
We have also determined that PBS-T (0.05 % in Tween-20) as the optimal buffer, which will allow proper bead migration balanced with an adequate conditions for DNA interaction and sample stability.
Aptamer Conjugation to latex beads
To attach our capture aptamers to latex beads, we have based on the previous work of INSA-Lyon 2016 iGem team [19],[20]. Our solution for aptamer attachment has been EDC crosslinking via carboxyl groups activation, and reaction with free amino-terminal groups of amino-modified aptamers.
We have tested several conjugation protocols, since many of them yielded poor beads migration results in the test membranes. Conjugation1 (adaptation of INSA-Lyon 2016 protocol) , Conjugation2 (adaptation of a EMD-Merck protocol for antibodies) and Conjugation3 (adaptation of a documented aptamer conjugation protocol) pool the three main protocols we have tested several times and optimized. Although the three protocols has been proved, just Conjugation3 have allowed our conjugated beads to flow through the analytical membrane.
Figures 11,12 and 13 summarizes the obtained experimental results trough the conjugation protocol optimization procedure. Cojugate1 protocol (using the described INSA-Lyon 2016 team protocol) have been tested with similar results to Conjugate2 protocol.
Figure 11.
Different 1% conjugated beads stocks at 1% following different conjugation protocols. a) Conjugate1 b) Conjugate2 c) Conjugate3. It is appreciable visible macroagregation of latex beads in the first tube, correspondent which the first trial of Conjugate1 protocol.
Figure 12.
Beads flow problems due to inadequate conjugation following conjugate2 protocol. Beads are resuspended in storage buffer. a) 1% bead suspension. b) 0.5% beads suspension. c) 1% beads stock in storage buffer. d) Blank with storage buffer. FF170HP Nitrocellulose membrane.
Figure 13.
Proper beads migration after conjugation following conjugate3 protocol. Beads are resuspended at 1% in PBS-T (0.05% Tween-20) buffer. a) FF80HP b) FF80HP blocked with BSA c) FF170HP d) FF170HP blocked with BSA e) FF80HP blocked with BSA, with directly conjugated beads resuspended in MES after 1h of conjugation.
Attending to the results depicted and the experience acquired during the experimentation we can obtain the following conclusions:
Working with latex beads as reporter particles is tricky, requiring a deep knowledge of their behavior during the conjugation and further handling steps. Our future plans will be replacing latex beads by gold nanoparticles or quantum dots. That sort of reporter particles has demonstrated to greatly enhance assay sensitivity, and there are well documented sources for proper handling. In further experimentation we will try to test them instead of latex beads.
Building the Lateral Flow Strips
LFA1 protocol sums up the procedure for building a semi-functionable strip sensor, and depicts how to test it with a working solution composed by aptamer conjugated latex beads incubated with E. Coli.
Figure 14 shows the results of the assay performed with different concentration of DNA test and control aptamers immobilized on the membrane.
Figure 14.
Half-strip assay after 15 minutes of elution. Different position of test and control regions, with 1x and 1:6 dilutions of the streptavidin incubated aptamers for detection (according protocol LFA1). a),b) 40 µM DNA-Streptavidin solution, with different T/C dot positions. c),d) 6.7 µM DNA-Streptavidin solution, with different T/C dot positions. There is no visible result.
As can be seen in the previous figure, there is no appreciable accumulation of latex beads in the T/C regions, it implies that the system has failed in some step of the detection system.
That absence of analytical signal can owe to three main reasons. Since we were running out of time in our project, we decided to make a deeper characterization of each possible fail-point.
A)
The Streptavidin is no affine enough for the nitrocellulose membrane we are using, leading to a diffusion trough the membrane. Immobilization of Streptavidin in narrow regions will be a requirement to avoid excessive displacement of the streptavidin.
B)
Our aptamer has not conjugated properly to the latex beads. The aptamer could have not been attached properly, or the affinity for the targeted molecule could have been lost after immobilization.
C)
Our selected pair of aptamers is not specific enough for the E. Coli DH5-alpha strain we are using.
Streptavidin stress test
Protocol Immob3 [17] details the experimentation for testing streptavidin as an anchoring system in membranes, during working conditions. Figure 15 depicts the results of the Stress test and the digital image processing of them.
Figure 15.
Streptavidin stress test results. 5 µg of streptavidin have been deposited in each dot, dripping two dots per membrane on different positions. a),c) Membranes sealed with microfluidic wax channels. b),d) raw unblocked membranes. e) Untreated membrane with 2.5 µg of streptavidin deposited in each dot. FF80HP membrane with 2.5 ug dots have not been displayed due to total absence of signal.
As figure 15 shows, is easy to appreciate how bigger pore sized membranes ease streptavidin displacement trough the membrane. In a) and b) assays (FF80HP membrane), the streptavidin dots has almost vanished and dispersed. In c),d) and e) assays (FF170HP membrane), the streptavidin has been better retained in the membrane, although it has also dispersed on an appreciable way.
It’s also remarkable that microfluidic channels helped in streptavidin retention, easing its concentration on the immobilization regions, the Figure 15 c) is a clear example of that. However, it’s also visible how streptavidin have eluted from the immobilization areas to the rest of the membrane.
We can conclude that though the observed affinity for nitrocellulose, streptavidin it’s not tightly attached to it. In order to guarantee enough concentration of the immobilized reagents via streptavidin, will be necessary to deposit the reagents on a very narrow area.
In order to improve the feasibility of lateral flow assay construction without requiring high skills or complex equipment, we have decided to employ synthethic biology as a solution. Inspire by INSA-Lyon 2016 team, we have posed the possibility to produce a cellulose binding domains modified streptavidin protein. More details about that part are detailed in our synthetic biology page.
Testing latex beads affinity.
Because our beads has not been able to show noticeable affinity in the membrane, we have designed an experiment to isolate latex beads interaction from other possible factors that may interfere during the lateral flow assay (high affinity of the complementary aptamer, membrane interactions or migration problems).
The Beads3 protocol details the experiment, whose results are showed in figure 16 and 17.
Figure 16.
Conjugated latex bead interaction assay. Test points consist of Coated 96 wells with Poli-T tail and E. coli as targets. Negative control has been made with BSA coated wells. Positive controls have been made in empty wells of a treated Nunc Maxisorp plate. a), g) INSA-Lyon 2016 conjugation protocol.b), h) 2-Step conjugation protocol. c), i) 1-Step conjugation protocol. d), j) Empty wells as blanks. e), k) Conjugated beads deposited in treated wells. f), l) BSA blocked wells for negative controls.
Figure 17.
Conjugated latex beads interaction assay. Relative absorbance difference compared to blank is depicted as signal of the assay. Test points consists on Coated 96 wells with Poli-T tail and E. Coli as targets. Negative control has been made with BSA coated wells. Positive assays has been performed in empty wells of an treated Nunc maxisorp plate. 2-Step(I) represents the conjugated beads following Conjugation1 protocol. 2-Step(II) represents the conjugated beads following Conjugation2 protocol. 1-Step represents the conjugated beads following Conjugation3 protocol.
Attending to the results offered by Figure 17, we can prove that our conjugated beads interact dimly with E. Coli and Poli-T tail targets. However, since the signal of BSA blocked wells is relatively high, compared with the signals of the assay, we can not assure that the interaction between beads and their targets is strong enough to keep them attached during the lateral flow assay.
We have also observed how all the prepared conjugated beads show slight affinity for their targets, which implies that at least a fraction of them have been functionalized with our capture aptamer.
It’s also noticeable that for all the conjugated particles, the interaction with Streptavidin-PoliT coated wells it’s more than a higher than for E. Coli coated well’s. That tendency itself implies that the results obtained with the three different conjugated beads suspensions are not a product of random error of the assay.
Finally we can conclude that our conjugation protocol has worked, and our conjugated beads are capable of recognizing their targets. However, the affinity they show it’s not strong enough to allow their direct utilization on a lateral flow format.
In any case, further experimentation is needed in order to elucidate which part is impeding the signal development in the assay.
Conclusions and Further Improvement
Summarizing all the knowledge acquired, we can conclude that aptamers are molecules with an enormous potential able to be applied in many different sensing technological approaches.
Lateral Flow Assay
On the other hand, the buildup process of a paper-based strip sensor has not been fully accomplished. However, we have established the basic workflow for developing the technology. We have studied and detailed step by step the basics principles of lateral flow assays. We hope that all the information we have gathered will be used in the future to improve the steps we have taken, ending on the creation of a valuable and versatile technological solution.
In our final lateral flow design, we have implemented two additional key aspects for device utility. Employing microfluidic for partial sample derivation and testing antibiotic resistance. As a proof of concept, we have planned to immobilize nitrocefin for B-lactamic antibiotic resistance [21]. Also, choosing as capture aptamer one targeting enterobacteria will allow performing multiplexed detection of different bacteria.
References
[1] Rowe, A., Bonham, A., White, R., Zimmer, M., Yadgar, R., Hobza, T., Honea, J., Ben-Yaacov, I. ad Plaxco, K. (2011). “CheapStat: An Open-Source, “Do-It-Yourself” Potentiostat for Analytical and Educational Applications”. PLoS ONE, [online] 6(9), p.e23783.
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