Exosomes play a vital role in intracellular communications and the exchange of substances. Compared with normal cells, tumor cells secrete more exosomes with tumor-specific proteins, which makes tumor-derived exosomes an important kind of cancer biomarker. Thus, the detection of tumor-derived exosomes can provide critical information for the diagnosis of cancer. However, the current detection methods for tumor-derived exosomes still have some shortcomings, including tedious operation and limited accuracy. It is necessary to develop a method with convenient operation and high sensitivity to detect exosomes. Surface-enhanced Raman spectroscopy (SERS) has been widely applied in the biological detection fields due to its excellent optical properties. SERS-based exosome detection methods have flourished in recent years. Many materials have been combined with SERS probes to achieve optimal detection results. Hydrogels are water-swellable polymeric materials with a three-dimensional (3D) network structure synthesized by crosslinking hydrophilic polymers. The porous structure of hydrogels is similar to that of the extracellular matrix. Specifically, acrydite-modified DNA can be easily incorporated into hydrogels during gel formation to recognize and immobilize biomolecules. More importantly, biomolecules can retain their intrinsic structure and function in hydrogels. Therefore, we wish to realize highly efficient and sensitive detection of tumor-derived exosomes by combining the SERS probe with hydrogels.

We demonstrate an optical detection of tumor-derived exosomes by developing SERS-active DNA functionalized hydrogels (denoted as SD hydrogels). The details of detection are presented in Fig. 1. SD hydrogels consist of two parts. One is SERS nanoprobes for the recognition of exosomes and the generation of SERS signals [Fig. 1(a)], and the other is DNA-functionalized polyacrylamide hydrogels (denoted as DPAAm hydrogels) for the immobilization of SERS nanoprobes and the amplification of Raman signals. These two parts are connected by the DNA in DPAAm hydrogels [Fig. 1(b)]. Figure 1(c) presents the detection principle of SD hydrogels for tumor-derived exosomes. Generally, SERS nanoprobes contain two recognition units, or in other words, one applies to all exosomes, and the other is only suitable for tumor-derived exosomes. Such an SD hydrogel takes advantage of SERS nanoprobes to distinguish the difference in the surface specific proteins between tumor and normal cells derived exosomes. Once tumor-derived exosomes appear, the interaction between SERS nanoprobes and DNA in DPAAm hydrogels is broken, followed by SERS nanoprobes falling from hydrogels with the help of PBS buffer, resulting in the weak SERS signals on account of the concentration of tumor-derived exosomes.

To obtain SERS probes (denoted as Janus ADD), Au NPs with about 3.5 nm diameter are modified by Raman reporter (DTNB) and recognition unit as DNA. The experimental results display that Janus ADD possesses a well-distinguishable Raman signal and has been functionalized with DNA (Figs. 2 and 3). Then, Janus ADD is immobilized into SD hydrogels by the acrydite-modified DNA aptamers. SEM image clearly demonstrates the porous structure of hydrogel [Fig. 4(a)]. The photographs indicate that SD hydrogels containing Janus ADD have been fabricated successfully. Subsequently, the features of SD hydrogels as SERS-active substrates are evaluated. The results show that SD hydrogels have the ability to amplify the Raman signals of Janus ADD, and the SERS signals at different points of SD hydrogels are homogeneous with a coefficient of variation of 6%. Besides, the SERS signals of three individual SD hydrogels have a relative standard deviation (RSD) value as low as 4%, which is of key importance for SERS sensors. Further, the detection ability of SD hydrogels is proved by the complementary aptamers at different concentrations ranging from 0 to 100 nmol/L in PBS solution. The SERS intensity of DTNB in SD hydrogels distinctly decreases with the increased concentration of complementary aptamers, indicating that SD hydrogels are suitable for biological detection. Finally, SD hydrogels are used to detect tumor-derived exosomes. SKBR3 exosomes are selected as a model and isolated from the cell media of SKBR3 cell lines. The obtained SKBR3 exosomes are consistent with the previous reports in vesicle structure and particle size. Moreover, SKBR3 exosomes can be observed in SD hydrogels by a super-resolution microscope. The concentration-dependent SERS intensity indicates that the SERS intensity decreases as the number of exosomes increases, and the SERS signals in target exosome groups are obviously much weaker than that of the blank control (Fig. 6). As a result, the limit of detection (LOD) of the present method is found to be approximately 22 μL^-1. The high sensitivity evidences that the SD hydrogels possess huge potential for the detection of tumor-derived exosomes in an easy and inexpensive manner at the point of care.

In this paper, SD hydrogels have been established to optically detect SKBR3-derived exosomes by immobilizing SERS nanoprobes into DNA-functionalized hydrogels. The SERS nanoprobes are used to recognize SKBR3-derived exosomes and generate fingerprint signals. DNA functionalized hydrogels serve a variety of functions, including providing a biocompatible environment for exosomes, supplying abundant sites for immune reaction, and amplifying Raman signals of SERS probes. The obtained SD hydrogel as a SERS active substrate has high uniformity, and the SERS signals obtained from DTNB by measuring at 1323 points have a coefficient of variation of 6%. Besides, the relative standard deviation of the SERS signal about DTNB in the three batches of SD hydrogels is about 4%. By taking advantage of the specific recognition ability and excellent Raman enhancement effect, the SD hydrogels are applied to the quantitative detection of SKBR3 exosomes with an ultralow LOD of about 22 μL^-1, which is two orders of magnitude lower than that of the conventional exosome detection methods. In view of the diversity of SERS probes, such an SD hydrogel is promising as a universal sensor for the detection of tumor-derived exosomes.