The development of methods for the detection of biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) is of great interest due to their crucial roles in biological processes. Existing methods for biothiol detection are invasive methods or poor penetration depth. To address these problems, magnetic resonance imaging (MRI) is a tool of choice for molecular imaging. 129xenon nuclear magnetic resonance (NMR) can be hyperpolarized by optical pumping to increase signal by up to four orders of magnitude. In addition, more than 200 ppm chemical shift window renders 129Xe NMR highly susceptible to the local chemical environment. Several 129Xe biosensors have been developed featuring a host molecular system encapsulating Xe and a targeting group for site-specific molecular recognition. Materials and Method We design a novel biosensor to detect biothiol through MRI. The biosensor incorporates two parts: cryptophane core and reactive group. Cryptophane-A, an ideal cage for the capture of xenon, was used for signal output. The acrylate group was selected to react with biothiols. This reaction-induced structural transformation of the 129Xe biosensor is expected to alter the electron density of the cryptophane cage, leading to a change in the 129Xe NMR chemical shift.We design a novel biosensor to detect biothiol through MRI. The biosensor incorporates two parts: cryptophane core and reactive group. Cryptophane-A, an ideal cage for the capture of xenon, was used for signal output. The acrylate group was selected to react with biothiols. This reaction-induced structural transformation of the 129Xe biosensor is expected to alter the electron density of the cryptophane cage, leading to a change in the 129Xe NMR chemical shift.The 129Xe NMR spectrum of biosensor shows two resonances that are assigned with dissolved free Xe at δ = 229.62 ppm and caged Xe in biosensor at δ = 76.87 ppm (Figure 1). A new signal at δ = 75.50 ppm appears within minutes upon addition of Cys. This new resonance is attributed to biosensor undergoing a thiol-addition reaction with Cys to produce a new cryptophane-A derivative. By recording 129Xe NMR spectra as a function of time (Figure 2), the intensity of the signal at δ = 76.87 ppm (Xe@1) gradually decreased in about 30 minutes. Concomitantly, the intensity of the new signal at δ = 75.50 ppm increased until it reached a plateau. Obviously, the chemical shift of caged Xe gives rise to a 1.4 ppm upfield shift after the addition of Cys. After addition of Hcy and GSH respectively, a similar 129Xe NMR spectral response was observed. The selectivity of biosensor for biothiols was evaluated. As shown in Figure 3, only biothiols caused the 129Xe chemical shift change. These results illustrate that biosensor has high selectivity toward biothiols and also can discriminate Cys from Hcy and GSH. We also investigated the property of biosensor for 129Xe MRI in vitro. As depicted in Figure 4, a frequency-selective gradient echo sequence centered at 75.50 ppm was used to obtain images of the biosensor. The result illustrates that Cys can be detected and localized by 129Xe MRI.we have presented a thiol-addition reaction based 129Xe biosensor for the detection of biothiols. The 129Xe biosensor is a simple functionalized cryptophane derivative, which is composed of cryptophane-A as host molecule for in-out Xe exchange and an acrylate group acting as a reaction site for the covalent binding to biothiols. This 129Xe biosensor shows good selectivity for biothiols among amino acids and analytes, and can discriminate Cys from Hcy and GSH.