key: cord-0322465-2rptfvwg authors: Urban, Nadine; Hörner, Maximillian; Weber, Wilfried; Dincer, Can title: OptoAssay – Light-controlled Dynamic Bioassay Using Optogenetic Switches date: 2021-11-08 journal: bioRxiv DOI: 10.1101/2021.11.06.467572 sha: ea4a9d56609d16ac46d5694b8c9da4c5235f2b5d doc_id: 322465 cord_uid: 2rptfvwg Circumventing the limitations of current bioassays, we introduce the first light-controlled assay, the OptoAssay, towards wash- and pump-free point-of-care diagnostics. Extending the capabilities of standard bioassays with light-dependent and reversible interaction of optogenetic switches, OptoAssays enable a bi-directional movement of assay components, only by changing the wavelength of light. Combined with smartphones, OptoAssays obviate the need for external flow control systems like pumps or valves and signal readout devices. The concept of the OptoAssay consists of two distinct areas ( Figure 1B) : a smaller receiver area where the signal readout is performed, is placed in the middle of the sender area into a notch so that the areas are not directly connected. These areas are later bridged by adding sample solution to both sides so that the assay components can diffuse from one area to the other. In this work, the OptoAssay is carried out as an immunoassay using antibodies. Herein, a competitive assay format was chosen, in which a competitor and the analyte in the sample compete for binding to the detection antibody. For this purpose, the sender area (Figure 1C) , on which the light sensitive components, i.e., PhyB, immobilized via a biotin-neutravidin (nAv) interaction on the surface is linked to PIF6 that can bind or be released to or from PhyB depending on the wavelength applied. On the other hand, antibodies directed against the molecule of interest are immobilized on the receiver area. For the proof-of-principle assay, we use a his-tagged protein (see Methods) as analyte and the his-tag on PIF6 as competitor that are recognized by an anti his-tag antibody. The signal readout is performed by measuring the fluorescence intensity of the competitor complex, his-tagged PIF6, to which green fluorescent protein (GFP) is fused. For the initial OptoAssay configuration, the competitor complex is attached to PhyB on the sender area during red light exposure. The assay procedure itself comprises of two steps ( Figure 1B) : first, the competitor complex is released from PhyB through far-red light illumination. At the same time, the analyte is added. Now both the analyte and the competitor can compete for binding to the antibody immobilized on the receiver area. Second, unbound competitor complexes are re-associated to PhyB present on the sender area and thus, removed from the receiver area only by applying red light. Due to the competitive assay format, the signal measured on the receiver area is inverse proportional to the concentration of the analyte. In order to enable POC testing using the OptoAssay, a 3D printed PhotoBox that allows for illumination with red and far-red light has been built (Figure1 D,E) 10 . The illumination can be controlled with a smartphone that is linked via a Bluetooth module to the electronics of the PhotoBox. Images of the OptoAssay results can be then easily read out, evaluated, and further transmitted using the smartphone. As substrate material for immobilization of assay components, we chose nitrocellulose as it is widely applied for LFA devices due to its non-specific affinity to proteins 11, 12 . We first investigated an appropriate blocking method for nitrocellulose to cover unoccupied spaces after the protein of interest has been immobilized. Therefore, we examined the blocking performance of bovine serum albumin (BSA) and casein. The results ( Figure S1 ) suggest that albeit casein has a better blocking performance, it seems to mask the previous immobilized proteins, rendering them inaccessible. Conclusively, we employed BSA as blocking agent for subsequent experiments. Since the PhyB/PIF interaction plays the key role in our experimental setup, we tested whether a competitor complex containing two PIF proteins shows better performance in terms of leakiness, i.e., fewer unspecific release during red-light illumination due to increased avidity. Therefore, the release of competitor complexes containing one or two PIF proteins was measured over a time range of 80 minutes. Additionally, to determine the unspecific release of the nitrocellulose membrane itself, the release of a biotinylated version of GFP-PIF6, immobilized directly on nAv, was determined. According to our findings (Figure S3) , the double PIF version has no advantages over the single version regarding the unspecific release (red light, 660 nm). In fact, the release of competitor molecules can be attributed to the unspecific release of the substrate itself as the biotin-GFP-PIF6 version shows similar values than the two other non-released samples. However, the specific release (far-red light, 740 nm) seems to achieve a higher release for the single PIF version, which is why we conducted all following experiments with this competitor version. In order to find an assay architecture in terms of the assay area and their positioning, we tested three different variants regarding the diffusion time and fluid distribution on the distinct areas ( Figure S2 ). For naked eye visualization, highly concentrated fluorescent protein mCherry (mCh) or a small molecule fluorescein was used. For both substances, the design variant where a small square shaped receiver area is enclosed by a larger square shaped sender area showed the best performance and was therefore employed for the following experiments For the proof-of-principle assay, we first demonstrated the light-induced competition of analyte and competitor. To simulate and verify the competitive mode of the OptoAssay, three conditions were tested: (i) red light illumination without analyte where no release of the competitor is expected, (ii) far-red light illumination without analyte where a release but no competition of analyte and competitor is possible, and (iii) far-red light illumination with analyte, where both a release and competition are expected. The fluorescence intensity measurements of the nitrocellulose membranes after illumination show a strong decrease in fluorescence signal of the sender membranes illuminated with far-red light compared to those illuminated with red light. Far-red light illuminated samples without analyte display a slightly higher fluorescence than the ones where analyte was added ( Figure 2A ). This can also be confirmed by measuring fluorescence intensities of the membranes ( Figure 2B) . Also, the measurements of the supernatant collected (Figure 1confirm this observation as there is a lower fluorescence intensity for the with far-red illuminated samples without analyte than the samples with. When comparing the leakiness (i.e., unspecific release of competitor during red light illumination) to the red-light induced specific release by dividing the fluorescence intensity of the farred by the red light illuminated sample, there is a 6.1-times increase in signals of the sample with analyte but only a 4.5-times signal increase for the sample without analyte. Since both samples were treated equally, except addition of an analyte protein, there is no obvious reason for those samples to behave differently. An explanation for this discrepancy might be a partial drying of the membrane after GFP-PIF6 addition and before illumination of the sample pair without analyte, which leads to PhyB denaturation and therefore, impairment of functionality. According to the fluorescence intensity of the receiver membranes, shown in Figure 2C , there was no negative effect on the competition itself; it is functional with even lower amounts of released competitor. While there is a high fluorescence intensity obtained from the sample treated with far-red light and without analyte, only a low fluorescence is detectable for the far-red light illuminated samples with analyte. A quantification of the fluorescence intensities of receiver membranes after illumination shows a 160% signal increase when comparing red light illuminated samples with far-red light illuminated ones without analyte and only a 20% increase in signals of far-red light illuminated membranes with analyte. This results in a fluorescent signal increase of 120% when comparing far-red light illuminated samples with and without analyte. The last step of the OptoAssay comprises of the rebinding of the unbound competitor molecules back to PhyB on the sender membrane which could enable a wash-free signal detection. To achieve this, one sample was illuminated first with far-red light to release the competitor and then with red light to rebind it. Control samples that were either illuminated with red or far-red light only were included as well. The fluorescence images of the membranes ( Figure 2D ) after illumination indicate that the control samples worked as anticipated. In the case of the sender membranes, the measured intensity of the far-red sample is lower than the red light one. Also, the binding of the competitor on the receiver membrane can be observed for the (far-red light illuminated) release samples ( Figure 2E ). The rebinding samples (illuminated with far-red and then red light) display a slight increase in fluorescence intensity of sender membranes. The rebinding of the unbound competitor was also demonstrated by measuring the supernatant fluorescence ( Figure 2F ) which corresponds the amount of free, unbound competitor. Here, the fluorescence intensity of the rebinding sample is only 0.6-times higher compared to the release sample. This means that about 40% of the released competitor could be rebound to the sender membrane. When comparing the membrane intensities ( Figure 2E ) before and after illumination, a reduction of fluorescence of the leakiness sample (red light) of 5% and 2% for sender and receiver, respectively, was observed. The release sample shows 36% reduction of intensity on the sender and a 23% increase on the receiver membrane. In the case of the rebinding sample, however, there is only a reduction of only 18% which indicates a partial rebinding. The receiver membranes prove only an increase of 7%. For this experiment, lower amounts of competitor were applied compared to the previous experiment to ensure a higher rebinding efficiency, as depicted in Figure S3 . The general lower fluorescence intensity of the rebinding samples, however, could be attributed to the longer incubation time of 80 minutes compared with 40 minutes used for the other samples. The longer incubation period might have led to higher dissociation of PhyB and PIF6 or even the unspecific release of nAv from the nitrocellulose membrane (see Figure S2 ). Having successfully demonstrated the proof-of-concept of the OptoAssay on a nitrocellulose substrate, we aimed to the test the general functionality of the optogenetic components on other substrates. As an alternative planar substrate, we used PMMA which is low-priced and thus, convenient for mass production of a potential POC device. A preliminary experiment ( Figure S5 ) showed that nAv directly absorbs to untreated PMMA which circumvents the need for a surface functionalization. Subsequently, we immobilized biotinylated PhyB on the nAv coated surface. With this setup, we could demonstrate a spatially resolved release of the competitor complex from a PMMA substrate ( Figure S6 ). However, we did not pursue with PMMA as a substrate because the total amount of immobilized photoreceptor was lower compared to nitrocellulose. The next substrate tested were functionalized beads that are very often used as solid phase materials in bioassay applications in clinical and POC diagnostics 13, 14 . In this setting, we verified the functionality of the optogenetic system on a bead-based platform where neutravidin (nAv) functionalized agarose beads are employed to immobilize the biotinylated PhyB. We simulated the reversibility of releasing and rebinding the PIF6 molecule as the competitor from PhyB as described in 9 , immobilized on agarose beads ( Figure S7 ). We could again show a light-dependent release as well as partial rebinding of competitor molecules from and to the substrate. Here, we have successfully demonstrated the proof-of-principle of an OptoAssay that enables, in contrast to conventional POC tests like LFAs, a bi-directional sample flow without any pump or flow control systems for a wash-free signal readout. Through a photoreceptor that can light-dependently interact with a binding partner, a competitor molecule can be released and later removed from the detection area by applying far-red and red light. Although other photoswitchable assays already exist 15, 16 , they only allow for detection of specific molecules, whereas the OptoAssay can be universally applied by fusing the molecule of interest to the phytochrome B interaction partner PIF6. For example, the integrating of a z domain 17 , an IgG-binding domain, antibodies can be attached to the PIF6 construct and act as detection antibodies to add even more versatility. The system could also be further expanded by using or combining different optogenetic switches that respond to distinct wavelengths. For instance, the blue light receptor cryptochrome 2 (Cry2) that forms heterodimers with its interaction partner cryptochrome-interacting basic helix-loop-helix (CIB1), or another switch, light-induced dimer (iLID), which comprises of the blue light receptor light oxygen voltage 2 domain of avena sativa phototropin 1-SsrA (AsLOV2) and its binding partner stringent starvation protein b (SspB) could be employed 18 . However, both photoreceptors only enable an active associating with its interaction partner, the dissociation is a slow passive event induced by the absence of blue light. Therefore, the OptoAssay workflow might need to be adapted accordingly in order to use different photoreceptors. Using a 3D printed PhotoBox along with a smartphone, the OptoAssay introduced can be employed for on-site applications. Herein, the PhotoBox could be extended by an excitation light and emission filter that allows for GFP detection using a smartphone. A low-cost and easy approach using paperbased filters in combination with a smartphone for a fluorescence readout has already been implemented 19 . Finally, automated sample illumination and evaluation of the results via a smartphone app could be realized for user friendly operation. Another issue that must be addressed is the overall operation time of the OptoAssay which is, at the moment, mainly limited by diffusion of the biomolecules employed. Herein, there are two important factors: the overall distance the molecules have to travel between the two areas (i.e., detection and immobilization zones) and the speed at which the molecules move. To decrease the distance, smaller assay areas could be designed and fabricated by micro/nanofabrication techniques. For increasing the speed, external energy, like ultrasonication, could be also applied to the system. The OptoAssay provides the basics for a novel and dynamic bioassay format (independent of assay type and biomolecules employed, such as antibodies, proteins or nucleic acids) through its light dependent two-way switch that can extend already existing POC devices and could pave the way for new classes of diagnostic devices in future, extending the capabilities of these state-of-the-art tools. The DNA sequences of the plasmids generated and used in this study are shown in Table 1 along with the templates and oligos for the polymerase chain reaction (PCR) to generate plasmids using Gibson cloning 20 . The sequences of the oligos used are listed in Table 2 . Figure 1D , 2B, 2E pMH1105 Plac/T7-phyB(1-651)-GSGS-AviTag-H6, Plac/T7-ho-pcyA 22 All optogenetic experiments pMH23 Plac/T7-zz-mcherry-pif6(1-100)-H6 Figure 1C pKJ10 Plac/T7-H6-mcherry Figure 1H pMH1409 Plac/T7-zz-mcherry-pif6(1-100)-GSS- For the buffer exchange from Ni-elution buffer to DPBS a 10 mL dextran desalting column (ThermoFisher Scientific, USA, 1896121) was used. The protein was aliquoted, shock frozen in liquid nitrogen and stored at -80 °C. The proteins were produced in bacterial strains of E. coli BL21 (DE3) (Invitrogen, USA). Cells were harvested by centrifugation at 6,500 g for 10 min and resuspended in lysis and disrupted using a French Cellulose sender areas were incubated in solution of a fluorescent protein mCh (molecular weight: 27,545 g mol -1 , concentration: 0.2 mM) or fluorescein (molecular weight: 332 g mol -1 , concentration: 10 mM) for 10 minutes. The treated sender and the untreated receiver membrane were assembled. Sender and receiver membranes were covered with DPBS, then images of the membranes were taken immediately after assembly and after 5, 10, 15, and 30 minutes. The Figure 2E with the same contrast settings as Figure 2B . Paper-based point-of-care testing in disease diagnostics Paper-Based Systems for Point-of-Care Biosensing Multiplexed Point-of-Care Testing -xPOCT Disposable Sensors in Diagnostics, Food, and Environmental Monitoring Phytochromes: An atomic perspective on photoactivation and signaling A light-switchable gene promoter system Spatiotemporal control of cell signalling using a light-switchable protein interaction Generic and reversible opto-trapping of biomolecules Enhanced Protein Immobilization on Polymers -A Plasma Surface Activation Study Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications Porous Bead-Based Diagnostic Platforms: Bridging the Gaps in Healthcare A Microflow Cytometry-Based Agglutination Immunoassay for Point-of-Care Quantitative Detection of SARS-CoV-2 Photoswitchable peptide-based 'on-off' biosensor for electrochemical detection and control of protein-protein interactions Quantitative Model for Reversibly Photoswitchable Sensors A synthetic IgG-binding domain based on staphylococcal protein a Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins Smartphone and Paper-Based Fluorescence Reader: A Do It Yourself Approach Enzymatic assembly of DNA molecules up to several hundred kilobases Synthetic Biology Makes Polymer Materials Count Production, Purification and Characterization of Recombinant Biotinylated Phytochrome B for Extracellular Optogenetics The authors would like to thank the Deutsche Forschungsgemeinschaft (DFG, German Research The authors declare no competing interests.