<i>In situ</i> construction of an SPR biosensor using cell-free protein synthesis technology and its application in drug screening for CXCR4

Xiaofei Wang; Yanting Wang; Hao Chen; Ningqi Xia; Jiayu Gu; Linfeng Zhang; Dongyao Wang; Bin Lu; Diya Lv; Xiaofei Chen; Yan Cao; Yifeng Chai

doi:10.1016/j.jpha.2026.101613

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Xiaofei Wang, Yanting Wang, Hao Chen, Ningqi Xia, Jiayu Gu, Linfeng Zhang, Dongyao Wang, Bin Lu, Diya Lv, Xiaofei Chen, Yan Cao, Yifeng Chai. In situ construction of an SPR biosensor using cell-free protein synthesis technology and its application in drug screening for CXCR4[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2026.101613

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Xiaofei Wang, Yanting Wang, Hao Chen, Ningqi Xia, Jiayu Gu, Linfeng Zhang, Dongyao Wang, Bin Lu, Diya Lv, Xiaofei Chen, Yan Cao, Yifeng Chai. In situ construction of an SPR biosensor using cell-free protein synthesis technology and its application in drug screening for CXCR4[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2026.101613

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In situ construction of an SPR biosensor using cell-free protein synthesis technology and its application in drug screening for CXCR4

doi: 10.1016/j.jpha.2026.101613

Xiaofei Wang^a,
Yanting Wang^a,
Hao Chen^a,
Ningqi Xia^a,
Jiayu Gu^a,
Linfeng Zhang^a,
Dongyao Wang^b,d,
Bin Lu^a,
Diya Lv^{c
,
,},
Xiaofei Chen^{c
,
,},
Yan Cao^{a,d
,
,},
Yifeng Chai^b,d

a. Department of Biochemical Pharmacy, College of Pharmacy, Naval Medical University, Shanghai, 200433, China;
b. Department of Pharmaceutical Analysis, College of Pharmacy, Naval Medical University, Shanghai, 200433, China;
c. Center for Instrumental Analysis, College of Pharmacy, Naval Medical University, Shanghai, 200433, China;
d. Shanghai Key Laboratory for Pharmaceutical Metabolite Research, College of Pharmacy, Naval Medical University, Shanghai, 200433, China

Funds:

This work was supported by the National Natural Science Foundation of China (Grant Nos.: 82473892 and 82174092), and the National Natural Science Foundation of Shanghai, China (Grant Nos.: 21ZR1483000 and 22ZR1476900).

Received Date: Apr. 29, 2025
Accepted Date: Mar. 24, 2026
Rev Recd Date: Mar. 23, 2026
Available Online: Mar. 25, 2026

Abstract

Abstract

Surface plasmon resonance (SPR) biosensor has emerged as a transformative tool in high-throughput drug screening and label-free analysis of biomolecular interactions. However, a critical limitation of SPR lies in its stringent requirement for highly purified proteins to ensure reliable quantification of binding affinities and kinetic parameters. In order to address the limitations, the lentiviral particle and styrene-maleic acid polymer have been previously developed to extract and stabilize transmembrane proteins (TMs) indirectly and thus to detect ligands interaction with TMs by SPR biosensor. The present study proposes a high-throughput SPR-based drug screening system that utilizes cell-free protein synthesis (CFPS) to achieve in situ purification and immobilization of TMs on SPR biosensors. First, C−X−C chemokine receptor 4 (CXCR4) protein with His-tag was prepared by CFPS. Then, two types of nickel-nitrilotriacetic acid (Ni-NTA) biosensors, modified with carboxymethylated dextran (CMD) and coated with polycarboxylate hydrogel coating (HC) matrix, were compared with classical carboxymethylated dextran 5 (CM5) biosensor in order to determine the optimal strategy for coupling the CXCR4-CFPS protein. The CMD-NTA/SPR biosensor was next applied to screen for CXCR4 ligands from 96 natural products. Finally, glycyrrhizic acid and ginsenoside Re were proved to be function of CXCR4 inhibitors by affinity test, molecular docking, and cell migration assay. The combination of CFPS with SPR technology facilitates in-situ purification and immobilization of target proteins in a single step, thereby significantly enhancing the efficiency of SPR assay procedures. The system has broad applicability for targeting various challenging TMs and provides potential candidates for subsequent drug development.
- Surface plasmon resonance,
- Cell-free protein synthesis,
- C−X−C chemokine receptor 4,
- In situ purification and coupling,
- High-throughput screening

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References(18)

References

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