Assembly of DNA-Templated Bioluminescent Modules for Amplified

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Assembly of DNA-Templated Bioluminescent Modules for Amplified Detection of Protein Biomarkers Yong Li, Panchun Yang, Ning Lei, Yihan Ma, Yaoting Ji, Chunnan Zhu, and Yun-Hua Wu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02734 • Publication Date (Web): 06 Sep 2018 Downloaded from http://pubs.acs.org on September 6, 2018

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Analytical Chemistry

Assembly of DNA-Templated Bioluminescent Modules for Amplified Detection of Protein Biomarkers Yong Li1,2*, Panchun Yang1, Ning Lei1, Yihan Ma3, Yaoting Ji2,4, Chunnan Zhu2, and Yunhua Wu1* 1

College of Life Sciences, 2Hubei Key Laboratory of Medical Information Analysis & Tumor Diagnosis and Treatment, College of Biomedical Engineering, and 3College of Chemistry and Materials Sciences, South-Central University for Nationalities, Wuhan, 430074, P. R. China. 4 Key Lab for Oral Biomedical Engineering of Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, 430079, P. R. China. ABSTRACT: By virtue of its self-illuminating mechanism, bioluminescence resonance energy transfer (BRET) technique has recently emerged as a promising platform for point-of-care (POC) diagnostics. However, due to the difficulty of incorporating generic affinity elements, such as aptamers and antibodies, current BRET-based methods are still not applicable to most of the clinically important biomarkers. Furthermore, the inability of these methods to amplify BRET signals leads to the limited sensitivity in some applications. Here, we present a modular strategy for amplified BRET detection of protein biomarkers in human peripheral blood samples. In this strategy, a DNA-templated bioluminescent module was constructed by simultaneously binding luciferase and green fluorescent protein to one DNA template in a site-specific manner. The proposed modules showed high energy transfer efficiency and could be assembled into the long self-illuminating polymers. Owing to this modular design, the aptamers and antibodies were rationally incorporated, enabling specific assembly of multiple bioluminescent modules on one target. This strategy realized the amplified BRET assays for human α-thrombin and prostate specific antigen (PSA) with the detection limit in picomolar range using either a spectrophotometer or a smartphone. The modularity of our strategy allowed detection of different biomarkers by simple exchange of affinity elements. Furthermore, the self-illumination and isothermal amplification performance of this strategy make it an attractive tool for POC diagnostics.

Bioluminescence resonance energy transfer (BRET) is a straightforward detection technique that involves nonradiative energy transfer from a luciferase to a suitable acceptor only when they are brought into close proximity and adjusted to favorable geometric orientation.1-5 In contrast to fluorescencebased techniques, especially the fluorescence resonance energy transfer (FRET), BRET is independent of external excitation, but utilizes the light emitted by luciferase as internal excitation energy. This self-illuminating mechanism avoids many problems associated with the external illumination of biological samples, including autofluorescence from samples, scattering of excitation light, photobleaching of fluorophores, and co-excitation of donor and acceptor.3,6-11 Furthermore, due to the omission of external light source, BRET assays can be simply performed with the portable devices, such as smartphone,8 paper-based device,12 and microfluidic system,13 which is especially suitable for pointof-care (POC) diagnostics in resource-limited settings. Based on these advantages, extensive efforts have been devoted to develop BRET-based methods for the detection of clinically relevant molecules.8,10,14 These methods are based on the simultaneous binding of luciferase and acceptor to one target by directly fusing or modifying them with the targetspecific affinity elements. The commonly used affinity elements in BRET assays mainly include receptors, peptides, and protein domains, which can interact specifically with a given target. Although the introduction of these affinity elements has realized detection of certain kinds of

biomolecules, current BRET-based methods are still not applicable to the majority of clinically important protein biomarkers. The main reason for this situation is the difficulty of incorporating more generic affinity elements into these methods, such as aptamers and antibodies. The direct modification of luciferase and acceptor with aptamers or antibodies usually causes the adverse effect on their optical properties. More importantly, the large molecular size of intact antibody may provide steric hindrance to significantly reduce energy transfer efficiency.3 Although the functional fragments of antibodies have been recently introduced into some BRET assays for small molecules,12 the incorporation of more widely used intact antibody is still challenging. In addition to the problem associated with the generality, the inability of present methods to amplify BRET signals results in the limited sensitivity and dynamic range in some applications. Therefore, it is highly desirable to develop a rational approach for incorporating more generic affinity elements into BRET assays, and further realize the amplified BRET detection of protein biomarkers. Recent development of the nucleic acid circuits have provided an emerging route to the design of biosensors and computing nanodevices for diagnostics.15-17 In particular, the hybridization chain reaction (HCR) has been proven to be a powerful signal amplification circuit.18-21 Based on the good compatibility of HCR circuit, the aptamers and antibodies can be incorporated for quantitative detection of various targets.18,22-24 Additionally, the HCR circuit can achieve rapid

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and efficient signal amplification under isothermal condition, which does not require large and expensive thermal cyclers, and thus meets the requirement of POC applications. Although, HCR circuit has been adapted to multiple detection modalities, such as fluorescence,25,26 electrochemistry,27-29 and 30,31 absorbance, it has not been applied to BRET assays so far. Integration of the self-illuminating mechanism of BRET and the benefits of HCR will enable the development of generic and sensitive BRET assays for different protein biomarkers. The key step of such a goal is to realize the specific recognition of luciferase and acceptor to the nucleic acid products of HCR circuit. In the present work, we designed a DNA-templated bioluminescent module in which the efficient energy transfer between luciferase and green fluorescent protein could be realized by simultaneously binding them onto one DNA template (Figure 1A). The proposed modules were efficient signal generating units that could be further assembled into the long self-illuminating polymers by a carefully-designed HCR circuit. Owing to the modularity of this system, the aptamers and antibodies could be easily incorporated, allowing the amplified BRET assays for human α-thrombin and prostate specific antigen (PSA) with the detection limit in picomolar range using either a spectrophotometer or a smartphone. EXPERIMENTAL SECTION BRET Signal Measurements and Recording. For the measurements of BRET signal, all samples were supplemented with an equal volume of Nano-Glo luciferase assay reagent. The emission spectra of resulting solutions were promptly measured using a spectrophotometer (F-2500, Hitachi) with the light source turned off. To record the BRET signals using the smartphone, the samples were transferred to a white 96 well plate (GREINER), and supplemented with Nano-Glo luciferase assay reagent. The emission of resultant solutions was recorded with the camera of iPhone 7 in a dark room. Preparation of Optimal DNA-Templated Bioluminescent Module. The optimal DNA-templated bioluminescent module was prepared by incubating 50 nM DNA template (spacer length = 20 ± 2 bp), 50 nM donor (nLZ-2), and 250 nM acceptor (mGA-1), in 50 µL of 1 × ZnK buffer composed of 20 mM Tris-Cl, 100 mM NaCl, 5 mM MgCl2, 0.1 mM ZnCl2, 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and 0.1 mg/mL bovine serum albumin (BSA) at pH = 7.4. After incubation for 30 min at room temperature, the samples were measured as described above. HCR and Detection of Initiator DNA. For detection of initiator DNA, the HCR reactions were performed by incubating 50 nM of each DNA hairpin (BH1 and BH2), 50 nM donor (nLZ-2), 250 nM acceptor (mGA-1), and various concentrations of initiator DNA in 100 µL of 1 × ZnK buffer for 30 min at room temperature. The BRET signals of resultant samples were measured as described above. To test the detection specificity, a mixture containing 0.1 µM of each mutant DNA target with 3-base mismatch, was used instead of perfectly matched initiator in control experiments following the procedure described above. Detection of Human α-Thrombin and PSA. The different concentrations of human α-thrombin were incubated with 0.5 mg/mL aptamer1-functionalized magnetic beads and 0.1 µM aptamer2-fused initiators for 30 min in 1 × phosphate buffered saline (PBS, pH = 7.2) or human serum. The resultant bead-α-

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thrombin-initiator complexes were separated with a magnetic scaffold, and washed 3 times using 200 µL of 1 × ZnK buffer. The washed beads were further mixed with 50 nM of each BH1 and BH2, 50 nM nLZ-2, and 250 nM mGA-1 in 100 µL of 1 × ZnK buffer. After incubation for 30 min at room temperature, the emission of each sample was analyzed as mentioned above. The detection of PSA was performed using antibody1functionalized magnetic beads, and antibody2-attached initiators. After incubating different concentrations of PSA with 0.5 mg/mL antibody1-functionalized magnetic beads and 0.1 µM antibody2-attached initiators for 30 min in 1 × PBS buffer (pH = 7.2) or human serum, the bead-PSA-initiator complexes were separated, washed, and analyzed following the same procedures as applied in α-thrombin detection. The detection specificity was verified by using 1 µM nonspecific proteins, which included human serum albumin (HSA), immunoglobulin (IgG), lysozyme, and trypsin. The condition of these control experiments was the same as that of α-thrombin and PSA detections. RESULTS AND DISCUSSION Construction of DNA-Templated Bioluminescent Module. We chose the NanoLuc luciferase (Nluc) and mNeonGreen as luminescent elements. The Nluc is an efficient luciferase due to its small size, good stability, and bright blue luminescence.32 Meanwhile, the mNeonGreen is a newly engineered monomeric green fluorescent protein with high extinction coefficient, quantum yield, and photostability.33 By construction of different sensor formats, the Nluc and mNeonGreen have been successfully employed by various BRET assays.8,34-36 Here, we sought to apply the benefits of Nluc and mNeonGreen to the design of DNAtemplated bioluminescent module.

Figure 1. The construction of DNA-templated bioluminescent module. (A) Schematic representation of modular configuration. The BRET between nLZ-2 and mGA-1 occurred only when they bind onto the DNA template containing both of Zif268 and AZP4 binding sites. (B) Bioluminescent spectra of the mixture consisting of nLZ-2 and mGA-1 in the absence (blue line) and presence (green line) of DNA template.

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Figure 2. Optimization of donor/acceptor pair of DNA-templated bioluminescent module. (A) Potential donors and acceptors were produced by respectively fusing Zif268 and AZP4 domain to each of Nluc and mNeonGreen at either terminus. (B) Construction of eight possible DNA-templated bioluminescent modules using different donor/acceptor pairs. (C) Emission ratios of different modules shown in (B). Error bars are standard deviation across three repetitive experiments.

To construct the DNA-templated bioluminescent module, the Nluc and mNeonGreen should be simultaneously bound onto one DNA template, and thus be brought into close proximity for efficient energy transfer. For this, two wellcharacterized zinc finger domains, Zif268 and AZP4,37,38 were respectively fused to the N-terminus of Nluc and C-terminus of mNeonGreen (Figures 1A). Because Zif268 and AZP4 can specifically bind to their cognate DNA sites with high affinity,39,40 the Zif268-fused Nluc (nLZ-2, as donor) and the AZP4-fused mNeonGreen (mGA-1, as acceptor) could simultaneously bind onto the double-stranded DNA template containing both of Zif268 and AZP4 binding sites (Figures 1A and S1). Such a binding process confined nLZ-2 and mGA-1 to one DNA template, which realized the efficient energy transfer between the two proteins (Figure 1B). The control samples without addition of DNA template only showed the donor emission (Figure 1B), confirming that the energy transfer between nLZ-2 and mGA-1 is the result of their binding to the DNA template. These results indicated that the DNA-templated bioluminescent module is successfully constructed by reasonably utilizing the specific recognition of two different zinc finger domains to one DNA template. Optimal Configuration of DNA-Templated Bioluminescent Module. The energy transfer efficiency depends on the relative orientation and distance between donor

and acceptor.1,5 Furthermore, the difference in the affinity of Zif268 and AZP4 toward their DNA binding sites may also affect the energy transfer efficiency. Therefore, the optimal performance of DNA-templated bioluminescent module can be achieved by adjusting these key configuration parameters. We first examined the effects of DNA binding affinity and relative orientation of donor and acceptor on the energy transfer efficiency. To this end, the Zif268 and AZP4 domain were respectively fused to each of Nluc and mNeonGreen at either terminus, which produced eight proteins as potential donors and acceptors (Figures 2A and S2). By combining these proteins into different donor/acceptor pairs, eight possible modules were further constructed (Figure 2B). All of these modules demonstrated BRET signals, which was due to the specific binding of donors and acceptors to the DNA template (Figure S3). However, different modules exhibited considerable variations in the mNeonGreen/Nluc emission ratio (Figure 2C). For modules using Zif268-fused Nluc as donors, the emission ratios were significantly higher than for the corresponding modules using AZP4-fused Nluc as donors. As previously reported, the DNA binding affinity of Zif268 is higher than that of AZP4.38,39 Because the binding of donor to the DNA template is an essential prerequisite for energy transfer, the

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higher binding affinity of Zif268-fused Nluc could lead to more efficient excitation of acceptor. Further comparison revealed that the highest emission ratio is realized by using nLZ-2 as donor and mGA-1 as acceptor (Figure 2C). These results confirmed that the nLZ-2/mGA-1 is the most efficient donor/acceptor pair for the construction of DNA-templated bioluminescent module. Because Zif268 and AZP4 bind in an "antiparallel" direction with respect to their DNA binding sites (N-terminus locates at 3' end of DNA sense strand),38,41 the Nluc domain of nLZ-2 and mNeonGreen domain of mGA-1 are confined closest relative to other donor/acceptor pairs (Figure 2B). Furthermore, this binding mode might also provide the favorable orientation between donor and acceptor. Having the optimal donor/acceptor pair, the effect of their distance on the energy transfer efficiency was further investigated. Since the nLZ-2 and mGA-1 were bound onto the DNA template, their distance could be adjusted by changing the length of spacer inserted between Zif268 and AZP4 binding sites (Figure 3). By screening different spacer lengths, the highest emission ratio was obtained with the spacer lengths of 20 ± 2 bp, which was determined to be the optimal distances between donor and acceptor (Figure 3).

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shown in Figure S5, the green lights emitted by the DNAtemplated bioluminescent module could be easily detected with the camera of a smartphone, indicating its potential application in the POC diagnostics. HCR-Mediated Assembly of Bioluminescent Modules. A challenge in the BRET assays is to realize the target-triggered signal amplification. The DNA-based design proposed above provides an opportunity for integrating BRET detection into the nucleic acid amplification circuits. Toward this goal, the HCR, an isothermal amplification circuit,18 was employed to assemble DNA-templated bioluminescent modules (Figure 4). The two kinetically trapped DNA hairpins (BH1 and BH2) were constructed by using single-stranded AZP4 and Zif268 binding sites as toeholds or loops, and 20 bp of doublestranded spacers as stems. The nLZ-2 and mGA-1 cannot bind to these monomeric hairpins, since the double-stranded DNA sites are required to the binding of Zif268 and AZP4 domain.37-41 Introduction of cognate initiator DNA triggers cross hybridization between BH1 and BH2, yielding the long linear DNA products, in which a large number of doublestranded AZP4 and Zif268 binding sites are formed and alternately arranged. These DNA products can be bound by nLZ-2 and mGA-1, which thus results in the assembly of bioluminescent modules into the self-illuminating polymers.

Figure 3. The emission ratios of different bioluminescent modules using the DNA templates with varied spacer lengths (0 bp to 40 bp). The nLZ-2/mGA-1 was the donor/acceptor pair. The shaded box highlights the range of optimal spacer length. Error bars are standard deviation across three repetitive experiments.

Together, the optimal modular configuration with high energy transfer efficiency could be designed by binding nLZ-2 and mGA-1 to the DNA template with the spacer length of 20 ± 2 bp. On the basis of this design, the molar ratio between donor and acceptor was further optimized. With a constant nLZ-2 and DNA template quantity, various concentrations of mGA-1 were titrated. The results showed that the emission ratio increases with the elevated mGA-1 concentration, and reaches a plateau when the mGA-1 is nearly 5-fold excess over nLZ-2 (Figure S4). Therefore, the optimal molar ratio of nLZ-2 to mGA-1 was fixed to be 1:5 throughout the subsequent experiments. Because the superior optical properties of Nluc and mNeonGreen are ideal for the POC applications in resourcelimited settings,8,12,42 we questioned whether the signal produced by the as-designed module is sensitive enough to be detected using the smartphone as a portable detector. As

Figure 4. Schematic representation of the HCR-mediated assembly of bioluminescent modules.

The feasibility of proposed HCR circuit was confirmed by the native polyacrylamide gel electrophoresis (PAGE), which showed that the formation of long linear DNA products with high molecular weight can be specifically triggered by the initiator (Figure S6). To verify the assembly of DNAtemplated bioluminescent modules, the nLZ-2 and mGA-1 were introduced into HCR circuit. As shown in Figure 5A, strong BRET signal was observed only when the hybridization between BH1 and BH2 was triggered by the initiator. These results suggested that the assembly of DNA-templated bioluminescent modules can be achieved by an initiatortriggered HCR circuit. Because the HCR is an efficient signal amplification technique for target detections,18,20 the initiator DNA can also be regarded as a target for evaluating the analytical

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performance of proposed system. Figure 5B showed a linear relationship between the emission ratios and the initiator concentrations. The detection limit was determined to be 5.6 pM with the linear range from 8 pM to 5 nM. Moreover, the amounts as low as 40 pM of initiator could be readily detected with the camera of a smartphone (Figure 5C), suggesting potential application of this system in the POC diagnostics. We next tested the specificity of proposed system by using mutant initiators with the mismatched sequences. The BRET signal in response to perfectly matched initiator was much higher than that in response to mutant one (Figure S7), which was consistent with the high specificity of HCR circuit.23,25,43 Therefore, the HCR-mediated assembly of bioluminescent modules is a sensitive and specific system, which rationally integrates the self-illuminating detection and isothermal signal amplification for the target analysis.

were employed as models because of their clinical relevance.44,45 As illustrated in Figure 6A, the proposed amplified BRET assay for human α-thrombin involves two steps: the immunomagnetic separation and HCR-mediated assembly of bioluminescent modules for signal amplification. Two αthrombin-specific aptamers were respectively attached to the magnetic beads (MBs) and fused with the initiator. Since these aptamers bind to the different epitopes of α-thrombin,46,47 the formation of sandwich complexes (MBs-α-thrombin-initiator) can be induced in the presence of α-thrombin, which was confirmed by the dynamic light scattering (DLS) and immunofluorescence analysis (Figure S8). After immunomagnetic separation, the sandwich complexes were mixed with the HCR hairpins, nLZ-2, and mGA-1, which triggered the assembly of multiple bioluminescent modules on one target, and thus produced amplified BRET signals.

Figure 6. Amplified BRET assay for human α-thrombin in 1 × PBS buffer. (A) Principle of α-thrombin assay that is performed using aptamers as affinity elements. (B) Linear relationship between the emission ratio and logarithm of α-thrombin concentration. Error bars are standard deviation across three repetitive experiments. (C) Image of BRET response to various concentrations of human α-thrombin. From left to right, the concentration of α-thrombin was 0, 14.4 pM, 72 pM, 0.36 nM, 1.8 nM, and 9 nM, respectively.

Figure 5. The signal amplification performance of HCR-mediated assembly of bioluminescent modules. (A) Bioluminescence spectra of the mixture consisting of nLZ-2, mGA-1, BH1, and BH2 in the absence (blue line) and presence (green line) of initiator DNA. (B) Linear relationship between the emission ratio and logarithm of initiator concentration. Error bars are standard deviation across three repetitive experiments. (C) Image of BRET response to various concentrations of initiator.

Amplified BRET Assays for Protein Biomarkers. Based on the modular design described above, we further attempted to expand our system to the amplified assays for protein biomarkers by incorporating aptamers and antibodies as generic affinity elements. The human α-thrombin and PSA

As shown in Figures 6B, it performed a linear relationship between the emission ratios and the concentrations of αthrombin. The detection limit of α-thrombin was calculated to be 12.8 pM with the linear range from 14.4 pM to 9 nM. In comparison with the unamplified BRET assay, which employed single bioluminescent module to directly detect αthrombin (Figure S9), the amplified BRET assay showed two orders of magnitude increase in sensitivity and a broader linear range, giving an amplification factor of 727 (Table S1). These results indicated that the target-triggered assembly of bioluminescent modules significantly improves the sensitivity of BRET assay. The reproducibility of proposed amplified BRET assay was evaluated by repeatedly measuring α-thrombin at five different concentrations (14.4 pM, 72 pM, 0.36 nM, 1.8 nM, and 9 nM) within the same day and within different days. The results showed that the coefficient variation (CV) of intra- and interassay was less than 6.0% and 9.0% respectively (Table S2),

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indicating an acceptable reproducibility for α-thrombin detection. The specificity of this assay was also examined by using four nonspecific proteins, including HSA, IgG, lysozyme, and trypsin. Strong BRET response was observed only when αthrombin was tested, whereas the BRET responses were negligible in the presence of nonspecific proteins (Figure S10). These results indicated that our method can be expanded to the amplified assays for a protein biomarker by incorporating aptamers as the affinity elements. In addition, the smartphonebased assay showed a similar picomolar detection limit as obtained using the spectrophotometer (Figure 6C), suggesting the applicability of this method to the POC detection of a protein biomarker. To demonstrate the generality of proposed method, the aptamers were replaced with antibodies as affinity elements for the detection of PSA. To the best of our knowledge, the BRET assay for PSA has not been reported previously. As shown in Figure 7A, two PSA-specific antibodies were attached to MBs and initiators through crosslinker and streptavidin-biotin interaction, respectively. Presence of PSA resulted in the formation of MBs-PSA-initiators sandwich complexes (Figure S11), which could trigger the assembly of DNA-templated bioluminescent modules for signal amplification. Based on this design, PSA could be detected with high specificity (Figure S12). The plots of the emission ratios versus the logarithm of PSA concentrations ranged from 11.2 pM to 7 nM showed a good linear relationship (Figure 7B). The detection limit of amplified BRET assay for PSA was determined to be 6.4 pM, which exhibited 3 orders of magnitude increase in sensitivity compared to the unamplified BRET assay using single bioluminescent module as signal generator (Figure S13 and Table S1). The amplification factor was therefore estimated to be 1484. Moreover, the intra- assay and inter-assay CV of presented method was lower than 5.5% and 9.5% respectively (Table S2).

Figure 7. Amplified BRET assay for PSA in 1 × PBS buffer. (A) Principle of PSA assay that is performed using antibodies as affinity elements. (B) Linear relationship between the emission ratio and logarithm of PSA concentration. Error bars are standard deviation across three repetitive experiments. (C) Image of BRET response to various concentrations of PSA. From left to right, the concentration of PSA was 0, 11.2 pM, 56 pM, 0.28 nM, 1.4 nM, and 7 nM, respectively.

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Importantly, the proposed amplified BRET assay for PSA could also be performed with the camera of a smartphone. The result showed that the picomolar concentrations of PSA could be readily distinguished from the control sample (Figure 7C). These results suggested that our method could be adapted to amplified assays for different proteins by simple exchange of affinity elements. Analysis of clinical samples. Having proved that the protein biomarkers could be detected in buffer solution, the analytical performance of proposed method in the complex sample was further investigated. As shown in Figure S14, the detection limit of α-thrombin and PSA in the spiked human serum was determined to be 18.3 and 7.7 pM respectively. The intra- and inter-assay CV was less than 6.5% and 9.5% in all cases (Table S2). The amplified BRET assay for these protein biomarkers in human serum could also be achieved by a smartphone. Compared with the results obtained in buffer solution, the analytical performance of our method was not significantly influenced by the complex components of human serum. This anti-interference ability could be attributed to the immunomagnetic separation of targets, which effectively removed nonspecific serum components and thus avoided some problems caused by these components, such as absorption of bioluminescent light and inhibition of luciferase activity.48 Therefore, the self-illumination of bioluminescence together with immunomagnetic separation provides an attractive format for the POC applications. Motivated by the good performance of proposed method in complex media, we next tested its capability for the analysis of clinical peripheral blood samples, which were obtained from Hubei Province Hospital of TCM. As shown in Table S3, the α-thrombin concentration in 10 blood samples determined by the present method showed an acceptable consistency with those obtained by the standard enzyme-linked immunosorbent assay (ELISA). The CV of intra- and inter-assay was less than 6.5% and 7.5% in all samples (Table S2). These results proved the feasibility of our method for the analysis of clinical samples.

Figure 8. Amplified BRET assay for PSA in peripheral blood samples. (A) The PSA levels in different samples. (B) Photograph of BRET response to the PSA levels in different samples.

The method was also applied to the detection of PSA in peripheral blood samples from 18 adult males. As shown in Figure 8A, the blood PSA levels in 6 samples were determined

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to be higher than normal cutoff value (4 ng/mL, equivalent to 0.11 nM), suggesting a high risk of prostate disorders for these people. These results were in satisfactory agreement with those of standard chemiluminescence immunoassay (CLIA, Table S4). Because the blood PSA level is the most widely recognized biomarker for early diagnosis of prostate disorders,49 the proposed amplified BRET assay provided a promising tool for discriminating patients with prostate diseases from healthy people. Moreover, the smartphonebased detection was also applied to the same peripheral blood samples described above. As shown in Figure 8B, the green light emission obtained from the 6 samples with elevated PSA levels can be easily distinguished from the blue-green light emission obtained from the samples with normal PSA levels. These results demonstrated the excellent performance of proposed method in the POC analysis of human peripheral blood. CONCLUSIONS In summary, we demonstrated a modular strategy for amplified BRET detection of protein biomarkers. The DNAtemplated bioluminescent module with high energy transfer efficiency was designed and optimized. By assembling these optimized modules into self-illuminating polymers, amplified BRET assays for α-thrombin and PSA could be performed using either a spectrophotometer or a smartphone. The modular design of this strategy makes it a versatile platform to be compatible with the generic affinity elements, including aptamers and antibodies. Therefore, the principle described herein has the potential to be expanded to other disease-related biomarkers, such as tumor cells, bacteria, and microRNAs. Furthermore, based on the self-illumination, isothermal amplification, and immunomagnetic separation of this strategy, the targets can be sensitively detected without sophisticated facilities, showing great promise for POC diagnostics in lowresource settings.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Chemical and reagents, supporting methods, supporting results and discussion, the sequence of DNA (Table S5), and the amino acid sequences of protein domains used in this study (PDF).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We are grateful to Mr. Wei Ni from Hubei Province Hospital of TCM for his help in collecting human peripheral blood samples. This work was supported by the National Natural Science Foundation of China (21405181; 31670372; 51502352), the

Fundamental Research Funds for the Central Universities (CZQ14017; CZP17072; CZZ18004), the Opening Project of Key Laboratory of Analytical Chemistry for Biology and Medicine (Wuhan University), Ministry of Education (ACBM2016009), and the Opening Project of Hubei Key Laboratory of Medical Information Analysis & Tumor Diagnosis and Treatment (PJS140011609).

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Figure 1. The construction of DNA-templated bioluminescent module. (A) Schematic representation of modular configuration. The BRET between nLZ-2 and mGA-1 occurred only when they bind onto the DNA template containing both of Zif268 and AZP4 binding sites. (B) Bioluminescent spectra of the mixture consisting of nLZ-2 and mGA-1 in the absence (blue line) and presence (green line) of DNA template. 66x77mm (300 x 300 DPI)



Figure 2. Optimization of donor/acceptor pair of DNA-templated bioluminescent module. (A) Potential donors and acceptors were produced by respectively fusing Zif268 and AZP4 domain to each of Nluc and mNeonGreen at either terminus. (B) Construction of eight possible DNA-templated bioluminescent modules using different donor/acceptor pairs. (C) Emission ratios of different modules shown in (B). Error bars are standard deviation across three repetitive experiments. 140x125mm (300 x 300 DPI)


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Figure 3. The emission ratios of different bioluminescent modules using the DNA templates with varied spacer lengths (0 bp to 40 bp). The nLZ-2/mGA-1 was the donor/acceptor pair. The shaded box highlights the range of optimal spacer length. Error bars are standard deviation across three repetitive experiments. 85x60mm (300 x 300 DPI)



Figure 4. Schematic representation of the HCR-mediated assembly of bioluminescent modules. 76x69mm (300 x 300 DPI)


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Figure 5. The signal amplification performance of HCR-mediated assembly of bioluminescent modules. (A) Bioluminescence spectra of the mixture consisting of nLZ-2, mGA-1, BH1, and BH2 in the absence (blue line) and presence (green line) of initiator DNA. (B) Linear relationship between the emission ratio and logarithm of initiator concentration. Error bars are standard deviation across three repetitive experiments. (C) Image of BRET response to various concentrations of initiator. 70x128mm (300 x 300 DPI)



Figure 6. Amplified BRET assay for human α-thrombin in 1 × PBS buffer. (A) Principle of α-thrombin assay that is performed using aptamers as affinity elements. (B) Linear relationship between the emission ratio and logarithm of α-thrombin concentration. Error bars are standard deviation across three repetitive experiments. (C) Image of BRET response to various concentrations of human α-thrombin. From left to right, the concentration of α-thrombin was 0, 14.4 pM, 72 pM, 0.36 nM, 1.8 nM, and 9 nM, respectively. 85x66mm (300 x 300 DPI)


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Figure 7. Amplified BRET assay for PSA in 1 × PBS buffer. (A) Principle of PSA assay that is performed using antibodies as affinity elements. (B) Linear relationship between the emission ratio and logarithm of PSA concentration. Error bars are standard deviation across three repetitive experiments. (C) Image of BRET response to various concentrations of PSA. From left to right, the concentration of PSA was 0, 11.2 pM, 56 pM, 0.28 nM, 1.4 nM, and 7 nM, respectively. 85x66mm (300 x 300 DPI)



Figure 8. Amplified BRET assay for PSA in peripheral blood samples. (A) The PSA levels in different samples. (B) Photograph of BRET response to the PSA levels in different samples. 70x66mm (300 x 300 DPI)


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Assembly of DNA-Templated Bioluminescent Modules for Amplified

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