Molecular interactions are a crucial aspect in chemical biology. In chemical biology research, small chemical molecules may either directly interact with targets or regulate the interactome between targets and other proteins. With the advancement of chemical biology, detection techniques for molecular interactions have also evolved, leading to the emergence of numerous methods with varying response speeds, precision, and application scopes. Regarding detection techniques for molecular interactions, please select the most accurate statement from the following options.
分子互作是化学生物学中的重要环节。在化学生物学研究中，化学小分子既可能与靶标存在直接相互作用，也可能调控靶标与其他蛋白质的相互作用组。伴随着化学生物学的发展，分子互作的检测技术同样推陈出新，涌现出了一大批具有不同响应速度、精度、适用范围的检测方法。关于分子互作的检测技术，请选出以下论述中最准确的一项。
Co-immunoprecipitation (Co-IP) is one of the most commonly used methods for detecting protein-protein interactions within cells, providing information about these interactions in the cellular context. Co-IP describes direct protein-protein interactions within cells and can be used for qualitative or semi-quantitative analysis. It is often combined with analytical methods such as mass spectrometry.
The proximity ligation assay (PLA) technology exhibits extremely high sensitivity, enabling the detection of extremely weak protein-protein interactions within cells. In the proximity ligation assay, DNA amplification can generate thousands of DNA strands; fluorescently labeled DNA fragments can complement the amplified products, thereby achieving signal amplification for weak signals. Additionally, when using the proximity ligation assay, it is important to avoid using primary antibodies from the same species to detect two interacting protein targets.
Microscale thermophoresis (MST) is a technique capable of detecting interactions without immobilization, offering advantages such as minimal sample requirement and rapid detection. MST specifically measures concentration changes caused by the thermophoresis of labeled molecules in a temperature gradient field, enabling the detection of molecular interactions in cell lysates. Considering a 1:1 binding process, when the receptor concentration is 50 nM, the fitted curve yields an $EC_{50}$ of 42.75 nM and a $K_d$ of 35.4 nM. The measured $K_d$ is slightly higher than the true value.
Isothermal titration calorimetry (ITC) can measure the thermodynamic effects of molecular interactions, binding stoichiometry, binding dissociation constant, and other information. ITC has strict requirements for the strength of molecular interactions. Assuming the binding constant of a small molecule compound to a protein is on the order of $10^{12}$, and the protein concentration is $10^{-7} mol· L^{-1}$, direct titration of the protein-small molecule interaction using ITC can only obtain the binding process $ΔH$, without information on $K_d$ or binding stoichiometry.
Surface plasmon resonance (SPR) technology is a method to measure interaction strength by detecting changes in the plasmon resonance angle and reflected light intensity before and after the binding of ligands to proteins immobilized on the chip surface. SPR technology can not only perform affinity analysis at the thermodynamic level but also characterize interactions at the kinetic level. Considering an SPR experiment with single-site binding and uniformly fixed receptors, the initial velocity of the adsorption process is usually faster, while the initial velocity of the desorption process is slower.
Biofilm Interference (BLI) technology is an emerging detection method. The principle of BLI is similar to SPR, but BLI measures the wavelength shift of the interference wave over time after binding. BLI has a broad monitoring range and is insensitive to buffer components, making it suitable for detecting various interactions, from proteins and small molecule inhibitors to antigen-antibody binding. It can even analyze interactions on the surface of live cells, but it cannot be used to estimate the concentration of specific biomolecules in crude samples.
免疫共沉淀（Co-IP)技术是最常用的检测细胞内蛋白-蛋白相互作用的方法之一，能够提供关于相互作用在细胞内的信息。免疫共沉淀描述的是细胞内蛋白-蛋白之间的直接相互作用，可以进行定性或半定量分析，并且经常与质谱等分析方法联用。
邻近连接（Proximal ligation assay）技术具有非常高灵敏度，可以检测在细胞内的极微弱蛋白-蛋白相互作用。在邻近连接技术中，通过DNA扩增可以产生成千上万条DNA链；具有荧光标记的DNA片段可以与扩增产物互补，从而实现微弱信号的放大。此外，使用邻近连接技术时需要注意不能使用来自同一物种的一抗检测两个互作的蛋白靶标。
微量热涌动（MST）技术是一种能够无需固定检测相互作用的方法，具有需要样品量少、检测快速等优势。MST专一地检测标记分子在温度梯度场内的热泳现象导致的浓度变化，可以用于对细胞裂解液中的分子互作进行检测。考虑一个1：1的结合过程，若在受体浓度为50nM时，拟合曲线两者的$EC_{50}$为42.75nM，$K_d$为35.4nM，则测得的$K_d$相比真实值略微偏大。
等温量热滴定（ITC）技术能够测量分子互作的热力学效应、结合计量比、结合解离常数等信息。ITC技术对分子互作的强度有严格的要求。假定小分子化合物与蛋白的结合常数的数量级约为$10^{12} $，蛋白的浓度为$10^{-7} mol· L^{-1}$，此时使用ITC对蛋白和小分子的相互作用直接进行滴定，只能获得结合过程$ΔH$，无法获得关于$K_d$和结合计量比的信息。
表面等离激元共振（SPR）技术是利用固定在芯片表面的蛋白在结合配体前后导致等离激元的共振角度变化和反射光的强度改变，来测量相互作用强度的方法。SPR技术不仅可以进行热力学层面的亲和力分析，还能对相互作用进行动力学层面的表征。考虑一个单位点结合且受体均一固定的SPR实验，通常吸附过程的初速度较快，而脱附过程的初速度较慢。
生物膜干涉（BLI）技术是一种新兴的检测方法。BLI原理与SPR接近，但BLI测量的是结合以后干涉波的波长位移随时间的变化情况。BLI具有宽广的监测范围，且对缓冲液组分不敏感，因此适用于从蛋白与小分子抑制剂到抗原与抗体结合等多种不同相互作用的检测，甚至可以分析活细胞表面的相互作用，但无法用于粗制样品中特定生物分子的浓度估算。
This question examines the description of the principles and application ranges of common interaction methods. Option A's statement "describing direct protein-protein interactions within cells" is incorrect because IP experiments often capture multiple proteins in protein complexes, where these proteins may not necessarily interact directly with each other. To prove direct interactions, additional experiments such as chemical crosslinking or in vitro pull-down assays are required, making this option incorrect.  

<Checkpoint>[Co-IP can detect either direct or indirect interactions](1)</Checkpoint>  

Option B describes PLA technology. PLA employs a rolling circle amplification method, which differs from traditional DNA replication by producing only a single DNA strand containing thousands of repeat fragments. These repeat fragments generate signals by hybridizing with fluorescent probes, making option B incorrect.  

<Checkpoint>[Rolling circle amplification in PLA technology produces only one DNA strand](1)</Checkpoint>  

Option C describes MST technology. MST not only detects thermophoresis but also measures temperature-dependent fluorescence intensity changes (TRIC) due to the presence of a temperature field. Therefore, the claim that it exclusively detects thermophoresis is incorrect. MST requires the receptor concentration to be lower than the $K_d$ value. In the given experiment, the receptor concentration is significantly higher, but the $K_d$ can still be estimated by fitting the Hill equation to obtain the $EC_{50}$. Since the binding is 1:1, the actual $K_d$ can be roughly estimated as $K_d = 42.75 - 0.5 \times 50 = 17.75 \, \text{nM}$, meaning the fitted result is correctly larger.  

<Checkpoint>[MST detects both thermophoresis and TRIC effects](1)</Checkpoint>  

Option D describes ITC technology. ITC has strict requirements for molecular interaction strengths, typically requiring the product of the binding constant $K_a$ and the initial protein concentration $K_a[P]_0$ to be between 10 and 1000. When $K_a[P]_0$ is less than 2, no information can be obtained. In the given system, $K_a[P]_0$ is approximately $10^5$. Under these conditions, although the exact $K_d$ cannot be measured directly, the ΔH and binding stoichiometry (n) can still be determined, making option D incorrect.  

<Checkpoint>[In the given ITC conditions, information about binding stoichiometry can be obtained](1)</Checkpoint>  

Option E describes SPR technology. For a single-site, uniformly fixed receptor SPR experiment, the interaction process can be described by first-order kinetics. Deriving the kinetic equations, the adsorption process yields $\frac{d[PL]}{dt} = c(k_{on}[L] + k_{off})e^{-(k_{on}[L] + k_{off})t}$, while the desorption process (with $[L] = 0$) gives $\frac{d[PL]}{dt} = ck_{off}e^{-k_{off}t}$. Considering the initial rate at $t = 0$, it is evident that the initial rate of adsorption is faster, making option E correct.  

<Checkpoint>[In SPR experiments following first-order kinetics, adsorption is typically faster initially while desorption is slower](1)</Checkpoint>  

Option F describes BLI technology, which is similar in principle to SPR. BLI is highly sensitive and widely applicable. Since its binding rate is often correlated with analyte concentration, it can determine the concentration of a specific molecule in a sample by fitting a standard curve. In practice, BLI is also commonly used to measure the concentration of carbohydrate compounds, making option F incorrect.  

<Checkpoint>[By fitting a concentration-binding rate standard curve, BLI can be used for concentration determination](1)</Checkpoint>
本题考察对于常见相互作用方法原理和应用范围的描述。A选项中“描述细胞内蛋白与蛋白直接相互作用”的论述错误，因为IP实验经常能够捕获蛋白复合物中的多种蛋白，此时这些蛋白彼此未必存在直接相互作用，若要证明存在直接相互作用，还需要补充化学交联、体外pull-down等实验，因此错误。

<Checkpoint>[Co-IP检测的既可能是直接相互作用也可能是间接相互作用](1)</Checkpoint>

B选项描述的是PLA技术，PLA技术采用的是滚环扩增方法，与传统的DNA复制不同，只会产生一条DNA链，但是含有成千上万个重复片段，这些重复片段与荧光探针互补而产生信号，因此B选项错误。

<Checkpoint>[PLA技术中的滚环扩增只能产生一条DNA链](1)</Checkpoint>

C选项描述的是MST技术。MST技术不仅检测热泳变化，由于温度场的存在还会检测到温度依赖的荧光强度变化（TRIC）效应的信号；因此专一地检测热泳效应的说法有误。MST要求受体浓度低于$K_d$数值，题设实验中，受体浓度明显偏高，但可以通过拟合Hill方程得到的$EC_{50}$估算$K_d$。由于是1：1结合，可以简单估算实际的$K_d = 42.75-0.5*50=17.75 nM$，因此拟合结果偏大是正确的。

<Checkpoint>[MST检测的既有热泳效应也有TRIC效应](1)</Checkpoint>

D选项描述的是ITC技术，ITC技术对分子互作强度有严格的要求，通常要求结合常数$K_a$与蛋白的初始浓度的乘积$K_a[P]_0$在10到1000之间，当$K_a[P]_0$小于2时，无法得到任何信息。题给体系中，$K_a[P]_0$约为$10^5$，此时直接滴定尽管不能测得准确的$K_d$，但仍然可以测得两者的ΔH和结合计量比n，因此D选项有误。

<Checkpoint>[题设ITC条件中可以获得关于结合计量比的信息](1)</Checkpoint>

E选项描述的是SPR技术。考虑单位点、受体固定均一的SPR实验，此时可以用一级反应描述两者的相互作用过程。推导动力学方程可以得到，吸附过程中$\frac{d[PL]}{dt} = c(k_{on}[L]+k_{off})e^{-(k_{on}[L]+k_{off})t}$，脱附过程中$[L]=0$，$\frac{d[PL]}{dt} = ck_{off}e^{-k_{off}t}$。考虑初速度则$t = 0$，此时不难看出吸附过程的初速度更快，因此E项正确。

<Checkpoint>[符合一级反应动力学的SPR实验中通常吸附的初速度快而脱附慢](1)</Checkpoint>

F选项描述的是BLI技术，BLI技术与SPR技术原理接近。BLI技术灵敏度高、适用范围广，且由于其结合速率通常与分析物浓度相关，因此可以通过拟合相应分析物的标准曲线来获取样品中某种分子的浓度信息。BLI在实践中也常被用于糖类化合物的浓度测定，因此F项不正确。

<Checkpoint>[通过拟合浓度-结合速率标曲，BLI可以用于测定浓度](1)</Checkpoint>