All on size-coded single bead set: a modular enrich-amplify-amplify strategy for attomolar level multi-immunoassay

Ultrasensitive protein analysis is of great significance for early diagnosis and biological studies. The core challenge is that many critical protein markers at extremely low aM to fM levels are difficult to accurately quantify because the target-induced weak signal may be easily masked by the surrounding background. Hence, we propose herein an ultrasensitive immunoassay based on a modular Single Bead Enrich-Amplify-Amplify (SBEAA) strategy. The highly efficient enrichment of targets on only a single bead (enrich) could confine the target-responsive signal output within a limited tiny space. Furthermore, a cascade tyramide signal amplification design enables remarkable in situ signal enhancement just affixed to the target. As a result, the efficient but space-confined fluorescence deposition on a single bead will significantly exceed the background and provide a wide dynamic range. Importantly, the SBEAA system can be modularly combined to meet different levels of clinical need regarding the detection sensitivity from aM to nM. Finally, a size-coded SBEAA set (SC-SBEAA) is also designed that allows ultrasensitive multi-immunoassay for rare samples in a single tube.


Preparation of mAb2-AuNPs-biotin DNA
The 16 nm colloidal AuNPs were prepared following the well-established citratereduction method. The mAb2-AuNPs-biotin DNA nanocomplexes were prepared according to a modified literature protocol. [S1] Typically, 1 mL of 16 nm colloidal AuNPs were adjusted to pH 9.2 -9.5 using a 0.1 M Na 2 CO 3 solution. Then, 10 μL anti-PSA mAb2 (1.0 mg/mL) was introduced, and the mixture was incubated at room temperature for 20 min with mild rolling. Afterward, the alkylthiol-capped 5'-Biotin oligonucleotides (1 nmol) were pipetted into the mixture. After gently shaking for 5 min, the mixture was further incubated at 4 °C overnight. Following that, the mixture was first buffered to 10 mM PB (pH 7.2), and then the NaCl concentration was tuned to 0.15 M under slow stirring. The mAb2-AuNPs-biotin DNA was purified by multiple rounds of centrifugation. Finally, the obtained mAb2-AuNPs-biotin DNA was dispersed in 1 mL of PBS-BSA and stored at 4 °C. The pre-treatment of the mAb2-AuNPs-biotin DNA with BSA as well as tween-20 can efficiently suppress their non-specific binding effect.

Preparation of capture antibody-conjugated SB
Typically, hundreds of SBs contained in 80 μL PBST were deposited onto a transparent 96-well plate cover hole to assist the capture of individual SBs. A micromanipulator system (Narishige) equipped on an Olympus IX53 inverted microscope with a monitor is used to manipulate a SB. With the help of the camera and monitor, we can search the SBs with suitable sizes on the screen with naked eyes.
A homemade ruler is used to help us select the beads with the desired narrow size (80 ± 5 μm). Once a desirable SB is selected, we only need a pipette to catch and transfer it into a tube. More detailed manipulations of pipetting microbeads have been described in our group's previous work. [S2,S3] We first select 20 uniform microbeads together in a tube, and add 100 μL ice-cold 1 mM HCl to activate their NHS surfaces.
Next, after isolation, add the mAb1 (0.1 μg) solution into the processed beads and mix well, and let the medium incubate with mild shaking for 1 h. Then the active site on the surface of the microbeads are effectively blocked by using Blocking buffer A and B in turn for three times according to the product manual. Then, we added 200 μL PBST to wash the microbeads (pipette and drain out the washing buffer PBST for 3 times and then the PBST is removed by magnetic separation), finally the mAb1conjugated microbeads are stored in PBS-BSA. Immediately before the immunoassay, the uniformly modified SBs are transfered into each reaction tube one by one.
It should be noted that since both the surfaces of mAb1-conjugated SBs and the mAb2/biotin-DNA-functionalized AuNPs are passivated with BSA, and BSA/Tween-20 are also added in the immunoreaction system, the nonspecific interaction between the SB and the AuNPs can be efficiently suppressed.

Standard procedures for the SBE strategy
The SB-mAb1 was incubated with 1 μL of PBS-BSA containing series dilutions of target antigen for 1 h. Moreover, 2 μL of biotinylated anti-PSA detective antibody (0.1mg/mL) was added to perform the noncompetitive sandwich immunoreaction at room temperature for another 1 h under mild shaking. After washing the superfluous detective antibody, 5 μL of excess AF546-STV (0.1 μg) in PBS was added for fluorescence staining. After 1h, the SB was washed and immediately subjected to fluorescence imaging.

Standard procedures for the SBEA strategy and SBEAA strategy.
The immunoreaction part of the SBEA strategy was the same as the SBE Strategy.
After washing the excess detective antibody, excess streptavidin Poly HRP (0.1 μg) was added into the bead. After 1 h incubation, wash the bead and prepare for TSA reaction.
The 10 μL TSA reaction solution was added to each SB and mild shake for 30 min.
After 1h, the SB was washed and immediately subjected to fluorescence imaging.
The only difference before TSA reaction between SBEAA strategy and SBEA strategy is replacing the biotinylated anti-PSA detective antibody with 2 μL mAb2-AuNPs-biotin DNA in the immunoreaction part. Since the sensitivity of SBEAA was further remarkably improved, we also optimized the reaction conditions for TSA reaction part of SBEAA. Finally for TSA amplification in SBEAA, mix 50 μL 1× TSA buffer and 2 μg biotin-tyramide together, then dilute the mixture 50 times by PBS, add 10 μL TSA reaction solution to each SB and mild shake for 30 min. The next steps are also the same as SBEA.
All fluorescence images were taken on an Olympus FV-1200 laser scanning confocal microscope following modified protocols in our previous report. [S2,S3] Briefly, the SB was spread on a coverslip, and its fluorescence image was obtained. By collecting the fluorescence at test parameters for AF546 of the instrument. The integrated fluorescence intensity of each SB was acquired for the quantitative analysis of the target antigen. It should be noted that the maximum fluorescence value of a bright spot that the fluorescence microscope can quantitatively acquire is 4096.
Therefore, to acquire a brighter MB image but not exceed this maximum value, the PMT voltage of the fluorescence microscope may be reasonably tuned for the imaging of MBs at different batches for different experimental conditions. 6. The original fluorescence imaging results of the SBEAA strategy for PSA detection Fig. S1 The original fluorescence imaging results of the SBEAA strategy for PSA detection. In order to display the results more intuitively, the corresponding pseudocolor results (displayed in different colors for different intensities) is showed in Fig. 2 in the main text.

Optimization of the concentration of TSA reagent for SBEA
According to the instruction of the commercial TSA Biotin Reagent Pack, the recommended TSA reagent for traditional biological staining is mixing 50 μL 1× TSA buffer (containing 0.01% H 2 O 2 ) and 1 μg biotin-tyramide together. Herein, to reduce the nonspecific background signal, so as to achieve the best signal-to-noise ratio in the SBEA, we further optimized the concentration of the reaction mixture on PSA detection. The reaction mixture was undiluted or was diluted 10 times, 50 times, 100 times, or 200 times with PBS buffer to detect 50 pg/mL PSA.
The concentration of TSA reagent has a significant impact on the single bead-based assay. The PSA-induced signal is strong under the high concentration of the reaction solution, while the corresponding blank value is also very high. Conversely, the sample signal is weak under the low concentration of the reaction solution, and the relative blank value is also low. However, due to the limitation of instrument range, it is impossible to accurately measure the fluorescence intensity of each group of experiments under the same test conditions. Therefore, we adjusted the PMT test voltage of the microscope to ensure that the integrated fluorescence intensities of each group of blank controls were almost the same. So that all test results were guaranteed within the detectable range. The difference between the signal intensity and the blank of the sample is then compared to evaluate the experimental conditions. The test voltage for undiluted, diluted 10 times, 50 times, 100 times, 200 times samples used in order: 350V, 380V, 410V, 460V, 480V. The experimental results are shown in Fig.   S2. It was found that when the TSA reaction mixture was diluted 10 times by the PBS buffer, the difference between the integrated fluorescence intensity of the sample signal and the blank signal was the maximum. Therefore, we finally determined that the TSA reaction mixture should be diluted 10 times with PBS buffer. So in the final reaction condition, tyramide concentration is 2 μg/mL with 0.001% H 2 O 2 .

Optimization of the TSA reaction time for SBEA
We also optimized the TSA reaction time. The experimental results at 10 min, 20 min, 30 min, 40 min, and 50 min of TSA reaction were tested. Likewise, we adjusted the test voltage so that the integrated fluorescence intensities of each group of blank controls was almost the same. The difference between the signal intensity and the blank of the sample is then compared to evaluate the experimental conditions. The test results are shown in Fig. S3. It can be seen that the difference between the experimental signal and the blank signal reaches the maximum at 30 min, so we chose the TSA reaction time of 30 min as the optimal condition for subsequent experiments.

The linear relationship between integrated fluorescence intensities (FI) of the beads and the logarithm of the PSA concentrations by using SBEA
We Integrate the fluorescence signals of each bead image for the quantitative analysis of the target PSA in the SBEA. As shown in Fig. S4, the beads' integrated fluorescence intensities (FI) are linearly proportional to the logarithm of the PSA concentrations in the range from 1 pg/mL to 100 ng/mL. The correlation equation is FI = 1.45 × 10 6 + 3.80 × 10 6 lgC PSA /(ng/mL), with a correlation coefficient (R) of 0.9973. Conjugating AFP antibodies on the 95 μm microbeads, 85 μm microbeads for CEA, and 75 μm microbeads for PSA, respectively. After such microbeads have been modified, blockeded, and washed respectively, one of each of the three types of microbeads is put into a single reaction tube together. After introduction of the samples containing different combinations of antigens, the three types of SBs can only enrich a correspondingly specific antigen after 1 h immunoreaction. Then, after further incubation with a cocktail solution containing three types of mAb2-AuNPsbiotin DNA for anoher 1 h, the three target-encoded SBs are subjected to TSA amplification and fluorescence imaging simultaneously. The following experimental procedures was the same as that for a single microbead-based SBEAA assay.

Evaluation of the specificity of the SBEAA
The specificity of the proposed SBEAA is interrogated by challenging

Evaluation of the generality of the SBEAA for the detection of CEA and AFP
We changed the types of antibodies conjugated with SB and AuNPs and examined the universality of SBEAA using CEA and AFP detection as an example. The results of CEA testing are shown in Fig. S6 and S7. The integrated fluorescence of SBs are proportional to the CEA concentrations in the range from 50 fg/mL to 50 pg/mL with the correlation equation FI = -1.69 × 10 6 + 6.10 × 10 6 lgC CEA /(fg/mL) and a correlation coefficient (R) of 0.9966.  Meanwhile, as shown in Fig. S8 and S9, AFP also showed similar test results.
There is a good linear relationship between the integrated fluorescence of SBs and logarithm of AFP concentrations from 10 fg/mL to 50 pg/mL with the correlation equation FI = 5.00 × 10 5 + 7.21 × 10 6 lgC AFP /(fg/mL) and a correlation coefficient (R) of 0.9900. These results demonstrate that by employing the corresponding target-specific antibodies, the proposed SBEAA can be readily extended as a general strategy for detecting various antigens.