AptaShield: A Universal Signal-Transduction System for Fast and High-Throughput Optical Molecular Biosensing

Biosensing technologies are often described to provide facile, sensitive, and minimally to noninvasive detection of molecular analytes across diverse scientific, environmental, and clinical diagnostic disciplines. However, commercialization has been very limited mostly due to the difficulty of biosensor reconfiguration for different analyte(s) and limited high-throughput capabilities. The immobilization of different biomolecular probes (e.g., antibodies, peptides, and aptamers) requires the sensor surface chemistry to be tailored to provide optimal probe coupling, orientation, and passivation and prevent nonspecific interactions. To overcome these challenges, here we report the development of a solution-phase biosensor consisting of an engineered aptamer, the AptaShield, capable of universally binding to any antigen recognition site (Fab’) of fluorescently labeled immunoglobulins (IgG) produced in rabbits. The resulting AptaShield biosensor relies on a low affinity dynamic equilibrium between the fluorescently tagged aptamer and IgG to generate a specific Förster resonance energy transfer (FRET) signal. As the analyte binds to the IgG, the AptaShield DNA aptamer–IgG complex dissociates, leading to an analyte concentration-dependent decrease of the FRET signal. The biosensor demonstrates high selectivity, specificity, and reproducibility for analyte quantification in different biological fluids (e.g., urine and blood serum) in a one-step and low sample volume (0.5–6.25 μL) format. The AptaShield provides a universal signal transduction mechanism as it can be coupled to different rabbit antibodies without the need for aptamer modification, therefore representing a robust high-throughput solution-phase technology suitable for point-of-care applications, overcoming the current limitations of gold standard enzyme-linked immunosorbent assays (ELISA) for molecular profiling.


Materials and Methods
Unless otherwise specified, all reagents were purchased from Sigma-Aldrich and used as received.
Furthermore, all buffers and aqueous solutions were prepared using ultrapure distilled deionized water (ddH2O) with a measured resistivity ≥18.0 MΩ•cm.The AptaShield DNA aptamer was synthesized by Integrated DNA Technologies (Leuven, Belgium).

Isothermal Titration Calorimetry
For isothermal titration calorimetry (ITC) experiments, the AptaShield DNA aptamer and IgG samples were exchanged into PBS buffer (10 mM phosphate buffer, 2.7 mM KCl and 137 mM NaCl, pH 7.4 at 25 °C.).ITC experiments were performed using a GE VP-ITC instrument.Data were fit to a one set of sites binding model using Origin 7 software.Samples were degassed prior to analysis using the GE ThermoVac unit.All experiments were acquired at 25°C and consisted of 29 successive injections 10 μL injections spaced every 400 s, where the first injection was 2 μL to account for diffusion from the syringe into the cell.Binding experiments were corrected for the heat of dilution of the titrant.AptaShield DNA aptamer titration experiments were conducted by titrating 80 μM of aptamer into 2.5 μM of IgG (rabbit anti-human Fc specific, purified rabbit IgG, or mouse anti-human IgG), with the AptaShield in the syringe and the IgG in the sample cell.

Rabbit IgG Fluorescence Labelling
Monoclonal rabbit anti-human IgG Fc-specific antibody was first exchanged into Bicarbonate reaction Buffer (2 mM NaHCO3, 100 mM NaCl, pH 8.3 at 25°C).The rabbit IgG was reacted with AlexaFluor 594 NHS Ester (ThermoFischer Scientific) at a 10x molar ratio in reaction buffer for 2 hours at RT.The reaction was stopped with ethanolamine at a 2x molar ratio of AlexaFluor 594 NHS in reaction buffer.The labelled rabbit IgG was then washed with PBS buffer (10 mM phosphate buffer, 2.7 mM KCl and 137 mM NaCl, pH 7.4 at 25 °C) using Millipore Amicon 20k centrifugal filters until three clear filtrates were achieved.

Fluorescence Labelling Efficiency
Fluorescence labelling efficiency was evaluated by UV-VIS spectrophotometry using a Nanodrop 1000 Spectrophotometer (Thermo Scientific).The UV-VIS spectra of a sample of ALEXA-594 rabbit IgG were acquired (230 -750 nm) using the appropriate molar extinction coefficients (Table S2) the concentration of IgG and ALEXA-594 were determined.The same procedure was used to confirm the fluorescence labelling of the AptaShield DNA aptamer with ALEXA-647 (Extinction Coefficients Table S1 and S2).

Titration of AptaShield DNA aptamer into Rabbit anti-Human IgG (and vice-versa) monitored by FRET
Different concentrations of ALEXA 647-AptaShield DNA aptamer or ALEXA 594 -rabbit anti-human IgG Fc-specific were titrated into 200 nM ALEXA 594 -rabbit anti-human IgG Fc-specific or 200 nM ALEXA 647 -AptaShield DNA aptamer respectively.The fluorescence emission spectra (Ex 550 nm) from 600 to 700 nm or just the emission intensities at 620 and 670 nm were acquired, using a Molecular Devices SpectraMax iD3.Binding curves related to AptaShield DNA aptamer/IgG titrations were produced by plotting the intensity ratios of 670/620 nm or 620/670 nm versus concentration of the titrant.Of note, the fluorescence intensity was background subtracted against the fluorescence signal obtained from the corresponding concentrations of 647 labelled AptaShield DNA aptamer.The apparent dissociation constants (Kd Apparent) were determined by fitting the binding curves to a one set of sites binding model using GraphPad Prism 8 software.

AptaShield Biosensor
Three proof-of-concept AptaShield biosensors were produced by mixing 150/50 nM, 300/50 nM and 600/50 nM of ALEXA 647 labelled AptaShield DNA aptamer and ALEXA 594 labelled Rabbit anti-human Fc-specific IgG (594-RAH).Purified human IgG Fc fragment (Abcam) was used as the analyte.To a 384 well plate format containing 18.75 μL of a specific AptaShield biosensor, 6.25 μL of purified human Fc spiked in either PBS, bovine urine (European Union Reference Material, ERM) or horse blood serum (Sigma Aldrich) was added.The fluorescence intensity was determined (Ex 550 nm, Emdonor 620 nm (ID), Emacceptor 670 nm (IA)) using a Molecular Devices SpectraMax iD3 and background subtracted against the signal from the corresponding concentration of ALEXA 647 labelled AptaShield DNA aptamer.FRET concentration response curves were constructed by plotting (ID/IA) versus concentration of analyte (purified human Fc).
The apparent dissociation constants (Kd apparent) were determined by plotting and fitting FRET -1 (IA/ID) concentration response curves to a one site binding model using GraphPad Prism 8 software.[3]

Enzyme-Linked Immunosorbent Assay (ELISA)
A 96-well plate (Nunc MaxiSorp) was coated with rabbit anti-human IgG Fc specific capture antibody (Sigma Aldrich) by incubation of 50 μL of 10μg/mL of antibody in immobilization buffer (0.1 M sodium carbonate/bicarbonate, 0.1 M NaCl, pH = 9.6) overnight at 4°C.The plate was washed 3x with TBS-T (0.01 M TRIS, 0.1 M NaCl, 0.05 % TWEEN-20, pH=7.4) and then blocked by incubation with 200 μL of 3% skim milk (ED Millipore) in TBS-T for 1hr at RT.The plate was washed 3x with TBS-T.For purified human Fc (Abcam) binding experiments, 100 μL of spiked PBS or ¼ diluted horse serum samples were incubated overnight at 4°C.The plate was then washed 3x with TBS-T followed by incubation of 100 μL of anti-human Fc-HRP labelled antibody diluted at a 1:25 000 ratio.The plate was washed 3x with TBS-T and incubated for 15 min with 100 μL 1x TMB substrate solution (Invitrogen)and then stopped with 100 μL TMB stop solution (Sigma Aldrich).The absorbance of the plate was read at 450 nm and background subtracted against the signal from wells that were incubated with analyte free PBS or ¼ diluted horse serum.The dissociation constant and concentration limit of detection were determined as described above.

Figure S1 :
Figure S1: Isothermal titration calorimetry titrations to determine heats of dilution of the titrant.

Figure S2 :
Figure S2: Repeated measurements over time of the fluorescence ratio (670/620 nm) of 300/50 AptaShield biosensor (uncorrected for AptaShield DNA aptamer-647 signal) and 50 nM RAH-594 in PBS, ¼ diluted bovine urine and ¼ diluted horse blood serum.The data shows that the ALEXA 594 on the RAH does not experience significant photobleaching and the fluorescence signal is stable over repeated measurements during a 15 minute time frame in PBS and a 10 minute time frame in urine and blood serum.The data is presented as mean ± SD with n=3.

Table S1 :
Sequence and physical properties of the AptaShield DNA aptamer

Table S2 :
Extinction coefficients of different reagents

Table S3 :
ITC derived thermodynamic and binding parameters of AptaShield DNA aptamer binding to IgGs.