Sandwich Immuno-RCA Assay with Single Molecule Counting Readout: The Importance of Biointerface Design

The analysis of low-abundance protein molecules in human serum is reported based on counting of the individual affinity-captured analyte on a solid sensor surface, yielding a readout format similar to digital assays. In this approach, a sandwich immunoassay with rolling circle amplification (RCA) is used for single molecule detection (SMD) through associating the target analyte with spatially distinct bright spots observed by fluorescence microscopy. The unspecific interaction of the target analyte and other immunoassay constituents with the sensor surface is of particular interest in this work, as it ultimately limits the performance of this assay. It is minimized by the design of the respective biointerface and thiol self-assembled monolayer with oligoethylene (OEG) head groups, and a poly[oligo(ethylene glycol) methacrylate] (pHOEGMA) antifouling polymer brush was used for the immobilization of the capture antibody (cAb) on the sensor surface. The assay relying on fluorescent postlabeling of long single-stranded DNA that are grafted from the detection antibody (dAb) by RCA was established with the help of combined surface plasmon resonance and surface plasmon-enhanced fluorescence monitoring of reaction kinetics. These techniques were employed for in situ measurements of conjugating of cAb to the sensor surface, tagging of short single-stranded DNA to dAb, affinity capture of the target analyte from the analyzed liquid sample, and the fluorescence readout of the RCA product. Through mitigation of adsorption of nontarget molecules on the sensor surface by tailoring of the antifouling biointerface, optimizing conjugation chemistry, and by implementing weak Coulombic repelling between dAb and the sensor surface, the limit of detection (LOD) of the assay was substantially improved. For the chosen interleukin–6 biomarker, SMD assay with LOD at a concentration of 4.3 fM was achieved for model (spiked) samples, and validation of the ability of detection of standard human serum samples is demonstrated.

The conjugation of biotin-dAb was performed in an in-situ step on the cAb modified carboxy-SAM by flowing streptavidin over the surface with four binding pockets, allowing the biotinylated primer sequence to bind, see Figure S2a.The click chemistry was done in a similar way, however the dAb was pre-modified ex-situ with DBCO-NHS ester, which could click to the azide tagged CS* in-situ (Figure S2b).As shown in Figure 3c, the maleimide-NHS ester was attached to the amine groups of the dAb and in a subsequent ex-situ step coupled to the sulfhydryl-activated CS* to form the dAb-CS* complex for the experiments on the biotin-SAM.

RCA reaction characterization by agarose gel-electrophoresis
The RCA reaction was performed in solution for 10 min, 20 min, 30 min and 1 h and afterwards loaded on an 0.8%-agarose gel for separation and visualization of the RCA generated DNA product.The amplification reaction was conducted with 20 µL of PL with molar concentration c = 40 nM, 2 µL of biotin/20T/TS* (40 nM), 1 µL of dNTPs (25 µM), φ29-Polymerase (20 units) and 13 µL NFW-BSA (0.2 mg/mL) on the HulaMixer at room temperature.After incubation of the indicated time, the reaction was stopped by inactivation of the enzyme on the thermomixer at 70°C and 700 rpm for 10 min.Then the DNA was loaded with 1:10 diluted loading dye into the pockets of 50 µL of the agarose gel for size separation at 100 V for 30 to 40 min.
Figure S3 shows the imaged gel with dark bands indicating the presence of the loaded DNA.Lane 1 and 2 show the 1 kilobasepair (kb) and 100 basepair (bp) ladder.However, after 10 min the RCA product already reaches a length, which can barely escape the loading pocket.With increasing RCA time this effect is even more pronounced.Lane 3 to 5 show the PL after ligation with molar concentrations of c = 40 nM, 4 nM and 0.4 nM in which the latter does not show a band due to low concentration.

SPR/PEF measurement on pHOEGMA-SAv brushes
The SA modified pHOEGMA-brushes were swollen in PBST and after 100 min coupled to biotin-cAb for the detection of IL-6 with molar concentration c = 48 nM.The maleimide coupled dAb-CS* complex was diluted in Tris-HCl buffer (pH 8.4) to which the PL could hybridize for the subsequent start of the RCA process for 60 min, monitored over time, shown in Figure S3a.Afterwards the generated ssDNA was labeled with Cy5-LS, producing a strong fluorescence signal ΔF = 1.0•10 5 cps, which is enhanced 1.7 and 2.7 times when contacting the surface with molar concentrations of CaCl2 c = 10 mM and 100 mM, as shown in the angular reflectivity R(θ) and fluorescence scans F(θ) in Figure S3b.The averaged fluorescence intensity plotted in Figure S4 was deduced from the images acquired by the confocal fluorescence microscope.The carboxy groups of the poly(HPMA-co-CBMA) brushes were prepared according to a previously published procedure and functionalized via the EDC/NHS coupling with cAb specific to IL-6.However, the surface was not contacted with any analyte, but tested for the unspecific response.To reduce the fluorescence signal originating from binding of assay constituents by active groups or the disruption of the neutral charge balance, the following deactivation agents with either sulfate/sulfo groups or carboxy groups were tested to deactivate the residual NHS esters on the surface: Tris-HCl (10 mM) with pH = 8.4, glycine (1 M) with pH = 7, spermine (20 mM) in HEPES buffer with pH = 8 and different ratios of glycine (G) to 2-aminoethyl hydrogen sulfate (D1) and aminomethanesulfonic acid (D2), see Figure S4a.Additionally, changing of the dilution buffer of the dAb-CS* complex for the deactivated surface with 96G:4D1 was tested (Figure S4b).Dependence on RCA reaction time Experiments have been performed according to the description in the 'Methods' section with the four-channel microfluidics mounted on the gold slide modified with the biotin-SAM.The analyte concentration was kept constant at c = 48 pM and the maleimide coupled dAb-CS* was diluted in Tris-HCl buffer with pH = 8.4.After the hybridization event of the PL, the RCA was conducted for 15 min, 30 min, 45 min and 60 min, respectively.For the control experiments for the same RCA timings, the surface was not in contact with the analyte.As depicted in Figure S5a, the average fluorescence intensity gradually increased with longer RCA running times.
Even the image after 15 min of RCA time showed significantly higher fluorescence signal F than the image of the control channel (see Figure 5b).

Serum samples
The experiments were performed as triplets on the pHOEGMA-SAv brushes with three different molar concentrations of IL-6 c = 2.89 pM, 930 fM and 70 fM and a control channel with c = 0 pM on one sensor slide with the microfluidic system and imaged after running the RCA for 30 min with the confocal fluorescence microscope.In Figure S6, the histograms of all three experiments are plotted, for which the threshold has to be determined individually.

S6
8. Specificity testing of biosensor Experiments were conducted in the 4-channel microfluidic system with fluorescence microscopy read-out for counting of number of spots N. As control of the specific capture of IL-6 from 1:10 diluted serum samples, the biotin-SAM surface was modified with either biotin-anti-TNF-alpha or biotin-BSA coupled via the neutravidin layer instead of the specific cAb to IL-6.After flowing the serum samples containing multiple potentially interfering biomolecules such as vascular endothelial growth factor, tumor necrosis factor alpha, epidermal growth factor, various interleukins, monocyte chemoattractant protein-1 and interferon gamma over the surface, the dAb-CS* dissolved in Tris-HCl (pH = 8.4) buffer and the PL were conducted according to previous experiments and the RCA was run for 30 min.

S7
9. Overview for immuno-RCA assays RCA already served as amplification technique in conjunction with immuno-assays for detection of different biomarkers.Table S1 provides an overview of the assays reported in literature and the stated analytical performance (limit of detection).
Table S1.Examples for immuno-RCA assays and the reported limit of detection for the specific analyte.

Figure S2 .
Figure S2.Schematical drawings for the conjugation of dAb to CS* via a) in-situ biotin and streptavidin bridge, b) in-situ click chemistry and c) ex-situ maleimide coupling.

Figure
Figure S4.a) R(t) in red and F(t) in blue of immuno-RCA on pHOEGMA-SAv brushes with cIL-6 = 48 nM and b) respective angular reflectivity R(θ) and fluorescence scans F(θ).

Figure S5 .
Figure S5.Plotted average fluorescence intensity acquired from experiments with immuno-RCA with running time of 60 min for IL-6 with c = 0 pM on poly(HPMA-co-CBMA) brushes testing a) different deactivation agents and b) buffer for dAb-CS* conjugate after deactivating residual NHS ester with 96G:4D1

Figure
Figure S6.a) Plotted average fluorescence intensity acquired from experiments with RCA running time of 15 min, 30 min, 45 min and 60 min with c = 48 pM in red and c = 0 pM of IL-6 in black and b) the respective images from the confocal fluorescence microscope.

Figure
Figure S7.a) -c) Plotted respective histograms of images acquired from the areas contacted with molar concentration of IL-6 of c = 2.89 pM, 930 fM, 70 fM and 0 pM from three different experiments.
Figure S8 shows the ratio of specific Na acquired from the serum samples with molar concentration of IL-6 c = 2.89 pM, 930 fM and 70 fM to unspecific response Nb with molar concentration of IL-6 c = 0 pM after setting the threshold to Ft = 1000 and counting of particles with ImageJ.

Figure S8 .
Figure S8.Specificity testing of immuno-RCA assay for IL-6 detection with serum samples of c = 2.89 pM, 930 fM and 70 fM on biotin-SAM biointerface modified with either anti-TNF-alpha or biotin-BSA as cAb.