组成图示
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传感器类型
表面等离子共振(SPR)生物传感器
检测对象
β-内酰胺酶（β-lactamase，TEM-1）；样品基质：PBS缓冲液、大肠杆菌粗细胞裂解液（crude cell lysate，含30–60 mg/mL非特异蛋白）
检测原理
3-MPA-(Ser)5-OH短肽SAM通过3-MPA硫醇端在金表面自组装，形成亲水抗污层，其C端羧基经EDC/NHS活化形成NHS酯，共价固定抗β-内酰胺酶抗体。目标TEM-1 β-内酰胺酶与抗体特异性结合后，在金膜表面约200–300 nm传感体积内增加质量并改变局部折射率。p偏振光激发金膜表面等离子体共振，共振波长λSPR随结合量发生移动。ΔλSPR在低浓度下与酶浓度线性相关，经PBS校准曲线定量；粗裂解液中先用空白裂解液条件化，SAM抑制非特异蛋白吸附，因此可直接读出特异性信号。该体系无外源标记和酶催化放大。
检测灵敏度
LOD: 约10 nM；线性范围: 44–700 nM；灵敏度斜率: 0.0025 nm/nM；R^2 = 0.991
效应效果
该传感器为无标记、无放大SPR检测，复杂基质抗干扰性好：3-MPA-(Ser)5-OH对未稀释牛血清非特异吸附132±33 ng/cm2，接近PEG单层100±27 ng/cm2，粗裂解液中较16-MHA降低约2倍；二次空白裂解液注入无额外信号。抗体固定ΔλSPR 8.35 nm，较NHS-MHA 2.15 nm提高4倍，活性抗体比例约87%。700 nM β-内酰胺酶响应1.8 nm，较NHS-MHA 0.13 nm和3-MPA-Gly-OH 0.25 nm提高约10倍。LOD约10 nM，与ELISA质量检出限相当。100倍稀释粗裂解液可直接定量，作者称与酶活法12.8 µM相符，支持临床耐药酶快速检测。
传感器的构成
- 基底/换能器：BK7玻璃片，溅射3 nm钛粘附层和50 nm金层，金膜作为SPR换能器，感应表面附近折射率变化
- 抗污连接层：3-MPA-(Ser)5-OH短肽自组装单分子层（SAM），3-巯基丙酸（3-MPA）硫醇端锚定金表面，C端羧基用于受体偶联，降低非特异蛋白吸附
- 化学活化层：EDC/NHS活化SAM末端羧基形成NHS酯，用于共价固定抗体
- 识别元件：抗TEM-1 β-内酰胺酶抗体（anti-β-lactamase antibody），特异性结合目标酶
- 失活/封闭剂：1 M乙醇胺盐酸盐（pH 8.5）灭活未反应NHS酯，终止偶联
- 信号读出：自研SPR仪器（dove prism波长 interrogation），实时监测SPR波长λSPR变化
中文摘要
短肽由极性或离子氨基酸与短有机硫醇组成，可显著降低血清和粗细胞裂解液等复杂生物基质中的非特异蛋白吸附，其中血清和粗细胞裂解液的非特异蛋白浓度分别为76和30–60 mg/mL。作者通过固相法合成不同链长和氨基酸组成的短肽库，将3-巯基丙酸（3-MPA）共价连接至肽的氨基端，形成3-MPA-(AA)n-OH并在金表面自组装成单分子层。接触角测量表明，极性或离子肽链长增加可提高金表面亲水性，而芳香和脂肪肽则降低亲水性。未稀释牛血清吸附实验显示，极性或离子氨基酸且链长n=5时非特异吸附最低。3-MPA-(Ser)5-OH单分子层具有显著优势：最小化未稀释牛血清非特异吸附、提供高表面抗体浓度、保留抗体活性、使β-内酰胺酶响应较先前实验提高约一个数量级，并允许在含30–60 mg/mL非特异蛋白的粗细胞裂解液中直接定量亚微摩尔β-内酰胺酶。该肽基单分子层为复杂生物介质中的定量SPR传感提供了重要优势。
英文摘要
Short peptides, composed of polar or ionic amino acids, derived with a short organic thiol, significantly reduce nonspecific adsorption of proteins in complex biological matrices such as serum and crude cell lysate, which have nonspecific protein concentrations of 76 and 30-60 mg/mL, respectively. Minimizing these nonspecific interactions has allowed rapid and direct quantification of beta-lactamase in a crude cell lysate using a surface plasmon resonance (SPR) biosensor. A library of short peptides with varying chain length and amino acid composition were synthesized using a solid-phase approach. A 3-mercaptopropionic acid (3-MPA) linker was covalently attached to the amino terminus of the peptides to subsequently form a monolayer on gold in the form of 3-MPA-(AA)(n)-OH, where n is the length of the amino acid chain (n = 2-5). Leu, Phe, Ser, Asp, and His were selected to investigate the effect on nonspecific adsorption with different physicochemical properties of the sidechains; aliphatic, aromatic, polar, acid, and base. Advancing contact angles measured the hydrophobicity of each peptidic self-assembled monolayer (SAM) and showed that hydrophilicity of the gold surface improved as the chain length of the polar or ionic peptides increased, while aromatic and aliphatic peptides decreased the hydrophilicity as the chain length increased. The nonspecific adsorption of undiluted bovine serum on SPR sensors prepared with the library of 3-MPA-(AA)(n)-OH showed that the lowest nonspecific adsorption occurred with polar or ionic amino acids with a chain length of n = 5. We demonstrate that a monolayer composed of 3-MPA-(Ser)(5)-OH has significant advantages, including the following: (1) it minimizes nonspecific adsorption in undiluted bovine serum; (2) it provides a high surface concentration of immobilized antibodies; (3) it shows a great retention of activity for the antibodies; (4) it improves the response from beta-lactamase by approximately 1 order of magnitude, compared to previous experiments; and (5) it allows direct quantification of submicromolar beta-lactamase concentration in a crude cell lysate with a nonspecific protein concentration of 30-60 mg/mL. The use of this peptide-based monolayer offers great advantages for quantitative SPR biosensing in complex biological media.