Purification of quantum dot-based bioprobes with a salting out strategy

A salting out strategy is reported for purification of IgG-conjugated QD (IgG-QD) bioprobes. Adding NaCl can precipitate free IgG selectively, while the IgG-QD maintains good colloidal stability. The dynamic light scattering technique reveals that this is due to the relatively positive zeta potential of free IgG than that of the IgG-QD.

a strategy under the mild conditions and facile process for the purication of QD-based bioprobes.
Salting out describes the precipitation of less soluble samples from a mixture solution aer adding electrolytes such as sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl 2 ), etc. 12 Aer two centuries of progress, scholars have employed salting out to separate chemical and biological samples such as proteins, polypeptides, DNA, etc. [13][14][15] Recently, Schroit et al. reported a salting out strategy to isolate tumor-derived exosomes with acetate by using charge neutralization. 16 Lee et al. developed a salting out strategy to sequentially separate graphene oxides by varying the ammonium sulfate concentrations. 17 Foster et al. reported that the yeast enzymes can be precipitated by using ammonium sulfate. 18 Gan et al. presented a salting out a strategy to isolate DNA from whole blood. 19 Ryall et al. reported a salting out strategy to precipitate the calcium oxalate in undiluted human urine using urate. 20 In our previous work, salting out with NaCl has been applied to separate the octylamine-graed poly-(acrylic acid) (OPA) micelles in OPA-coated QDs solution. 21 Herein, inspired by the above-mentioned studies, salting out is used to separate free Immunoglobulin G (IgG) in IgGconjugated QD (IgG-QD) solution. As illustrated in Fig. 1a, the addition of NaCl can preferentially compress the zeta potential of free IgG to electrical neutrality due to the relatively positive zeta potential of free IgG, resulting in the aggregation and precipitation of free IgG, while the IgG-QD still maintains good colloidal stability due to its relatively negative zeta potential. Therefore, the separation of free IgG in IgG-QD solution can be achieved effectively by adding an appropriate concentration of NaCl.
The hydrophobic CdSe/CdS QDs were prepared by following our previous method. 8,21 As shown in Fig. 1b, the transmission electron microscopy (TEM) image of the as-prepared QDs illustrated their uniform diameter of 7.3 AE 1.2 nm (Fig. S1 †). Subsequently, OPA was synthesized (Fig. S2 †) and utilized to prepare hydrophilic OPA-coated QDs (OPA-QDs). As shown in Fig. S3 water solution aer adding OPA, indicating that the hydrophilic OPA-QDs were prepared successfully. Aer that, the OPA-QDs were puried with NaCl to remove the OPA micelles according to our previously reported strategy. 21 The TEM picture of the puried OPA-QDs displayed in Fig. 1c shows that with the assistance of phosphotungstic acid (1%), no empty OPA micelles can be observed in the NaCl-treated OPA-QDs, suggesting that the OPA micelles were separated from the OPA-QD solution successfully. The photoluminescence (PL) spectrum showed that the as-prepared OPA-QDs emit red uorescence at 628 nm (Fig. 1d). Then, amine-PEG-carboxyl (NH 2 -PEG-COOH, MW ¼ 2000) reacted with the carboxyl of OPA-QDs to obtain OPA-QDs-PEG for the further bioconjugation and the decrease of non-specic adsorption. As shown in Fig. S4, † aer reacting with NH 2 -PEG-COOH, the electrophoretic speed of OPA-QDs-PEG is signicantly slower than that of OPA-QDs, indicating that NH 2 -PEG-COOH was graed on OPA-QDs successfully. Next, the resulting OPA-QDs-PEG (3 mmol, 200 mL) was conjugated to 1 mg of IgG (anti-mouse second antibody, 1 mL) to obtain the IgG-QD probes. Subsequently, we added 1 mL of NaCl (2 mol L À1 ) into IgG-QD (3 mmol L À1 , 200 mL) solution, and the IgG-QD solution (Fig. S5, † le) became turbid rapidly (Fig. S5, † middle) and also could be changed to transparent again aer 2 h (Fig. S5, † right). This is in agreement with our previous results that the turbid NaCl-treated OPA-QD solution can be recovered to transparent again, indicating that the free IgG can be precipitated selectively from IgG-QD solution aer adding NaCl. The purication results were characterized by SEC. As can be seen in Fig. 2a-c, the retention time of free IgG (Fig. 2a) and IgG-QD (Fig. 2b) is 33 min and 24 min, respectively. Aer salting out with NaCl, the chromatographic peak of the free IgG is disappeared for the IgG-QD solution (Fig. 2c), suggesting that the free IgG was removed from the IgG-QD solution. It should be noted that a high IgG to IgG-QD ratio and the electrolyte with an appropriate type and concentration are benecial for the selective precipitation of free IgG from IgG-QD solution.
The puried IgG-QD was collected and dispersed in ultrapure water by using a centrifugal lter device to remove NaCl for the dynamic light scattering (DLS) measurements. The zeta potential of IgG and IgG-QD probes was investigated by DLS before and aer adding NaCl. As shown in Fig. 2d, aer the addition of NaCl (2 mol L À1 ), the zeta potential of free IgG increased from À12 mV (Fig. 2d, sample 1) to ca. 0 (Fig. 2d, sample 2) and can be recovered to À13 mV (Fig. 2d, sample 3) aer removing NaCl with the centrifugal lter device. 22 Similarly, IgG-QD probes increased from À32 mV (Fig. 2d, sample 4) to À16 mV (Fig. 2d, sample 5), and can also return to À30 mV (Fig. 2d, sample 6) aer NaCl is removed. 23 The results of Fig. 2d suggested that the free IgG can be preferentially compressed to electrical neutrality due to the zeta potential of free IgG being closer to zero than that of IgG-QD probes aer the addition of electrolytes, and the hydration layer of the free IgG can also be destroyed which can induce the aggregation and selective precipitation of free IgG from IgG-QD solution. However, IgG-QD probes still maintain good colloidal stability under the identical salting out conditions due to their relatively negative zeta potential. Subsequently, gel electrophoresis was utilized to characterize the IgG-QD probes before and aer NaCl treatment. As shown in Fig. S6, † the electrophoretic speed of NaCl-treated IgG-QD solution is slower than that of IgG-QD probes noticeably, indicating that the surface charge of IgG-QD probes can be increased nearly to zero with the addition of NaCl, which is in agreement with our previous results. 21 Based on the above results and our previous reports, we assert that the zeta potential of both the free IgG and IgG-QD probes is compressed simultaneously, while IgG with a relatively positive zeta potential is preferentially compressed to electrical neutrality and is precipitated, thus the separation of free IgG in IgG-QD solution is achieved through adding NaCl. Based on the previously reported studies, 24 we speculate that other electrolytes (e.g. KCl and (NH 4 ) 2 SO 4 ) should also be used for the purication.  The optical properties of IgG-QD solution were also examined before and aer treating with NaCl solution. As shown in Fig. S7, † both the absorption and PL spectra of puried QDs, OPA-QDs and IgG-QD exhibited identical proles before and aer being treated with NaCl, indicating that the integrity of QDs was maintained in the process of salting out. Moreover, adding NaCl produced a negligible inuence on the quantum yield (QY) (Fig. S8 †) compared with that of OPA-QD solution, suggesting that the surface structure of QDs was maintained compared with that of original samples.
Next, the IgG-QD with green PL emission was prepared and utilized to recognize the cytokeratin 8/18 (CK8/18) antigens which are a member of the cytokeratin family, and their expressions are associated with breast cancer and are utilized to monitor the treatment process in breast cancer. 25 The redemission IgG-QD was then utilized to recognize the P63 antigens which are a member of the p53 gene family, and their germline mutations are associated with human mammary cancer. 26 As illustrated in Fig. 3a and c, green uorescence was localized in the cytomembrane, which is consistent with the previous reports, suggesting that the CK8/18 antigens are expressed in the cytomembrane. 27 As illustrated in Fig. 3b and d, red uorescence was localized in the cell nucleus, which is consistent with the previous results, 28 suggesting that the salting out process exhibits a negligible inuence on the target recognition of QD-probes.
Compared with the conventional purication strategy of IgG-QD bioprobes, three main advantages of our salting out strategy are to be highlighted. First, the purication of IgG-QD bioprobes can be achieved only by adding an appropriate amount of NaCl solution, thus the puried process is time-and laborsaving. Second, no supplementary instruments, such as the high performance liquid chromatograph and ultracentrifuge, are required, thus the purication can be realized in a general laboratory. Last and the most important, the biological function of QD-based probes can be maintained aer purication because of the mild purication conditions without high pressure and high speed centrifugation, which is benecial for maintaining the target recognition of the IgG-QD.
In conclusion, a purication strategy of IgG-QD bioprobe solution containing free IgG is presented based on a "charge neutralization" strategy. Aer treating NaCl, the free IgG can be aggregated and precipitated from IgG-QD solution. Using zeta potential analytical techniques, we have revealed that by adding NaCl, the relatively positive zeta potential of free IgG is compressed to electrical neutrality preferentially, which can induce the aggregation and precipitation of free IgG, while IgG-QD maintained good colloidal stability due to its lower zeta potential than that of free IgG. Moreover, the optical properties, target recognition, and colloidal stability of the puried IgG-QD are maintained aer salting out. This salting out strategy is suitable for the purication of the free IgG in IgG-QD solutions. This work facilitates the purication of QD-based bioprobes, and will contribute to the isolation of other impurities, such as the DNA or polypeptide in the bio-functionalization process of nanoparticles.

Conflicts of interest
There are no conicts to declare.