Tumor cell pH detection based on CdSe quantum dots’ fluorescence charateristics

2.1Materials

Cadmium oxide (CdO, AR), stearic acid (98%), (OA, 99%), selenium powder, octylamine, HAD and diethylzinc (ZnEt2) (Aladdin), TOPO (98%), TOP (95%), poly (acrylic acid) (PAA, molecular weight (MW) 1,800), 1-ethyl-3-[3-dimethylaminoporpyl] carbodiimide hydrochloride (EDC, 98.5%), NHS (98%), Bovine serum albumin (MP Biomedicals Company), Bis sulfide (Tokyo Chemical Industry), Liquid paraffin, chloroform, ethanol, hydrochloric acid (HCl), 2-(4-morpholino) ethanesulfonic acid, Ndimethylformamide, paraformaldehyde, Tween-20 (Sinopharm Chemical Reagent).

2.2Fluorescence spectrophotometer

For the fluorescence detection device, we use the fluorescence spectrophotometer for fluorescence testing. The fluorescence spectrophotometer is Hitachi FL-4600. The detection device has high precision and high sensitivity.

2.3Experimental method

2.3.2pH test method

For each test, mix 2 μL of the quantum dot with 298 μL of PBS buffer (0.2 mol/L) of different pH values and mix them up in a small test tube. Then use tin foil to keep light away. As shown in Fig. 1, after thoroughly mixed, use fluorescent test device to test.

Figure 1.

Quantum dot mixture to be tested.

After sufficient reaction, transfer solution to the slit cuvette. The cuvette is placed in the fluorescence test apparatus and continuously irradiated for 10 min by 365 nm ultraviolet laser. The excitation light wavelength is 365 nm. Read data each 1 minute and test for 10 minutes. Finally, record the fluorescence intensity.

3.1Fluorescence data processing

The wavelength of light absorbed by CdSe quantum dots in paper is 365 nm and the wavelength of the fluorescence spectrum is about 625 nm. The fluorescence spectrum of the quantum dots is analyzed by using the fluorescence detection device. The synthesized quantum dots show strong fluorescence in Fig. 2. With the signal and narrow emission spectra, CdSe QDs synthesized by this method have narrow particle and normal size.

Figure 2.

Quantum dots fluorescence spectroscopy.

Figure 3.

Moving average filtered fluorescence spectra of quantum dots.

Because the quantum dot fluorescence intensity at 625 nm is the largest and the data is the most accurate and easy to analyze, the fluorescence intensity data used in this test is derived from the fluorescence intensity data at 625 nm in the QD photoluminescence spectrum.

Because fluorescence spectrum obtained by fluorescence detection device still has noise, to obtain a more accurate fluorescence emission peak of the quantum dot, use a moving average filter to perform filtering to obtain the emission peak of the quantum dot accurately and the result is shown in Fig. 3.

3.2pH results

The raw data for the 10 minutes test is shown in Table 1.

The average value of test data for 10 minutes is calculated as the fluorescence intensity at the corresponding pH value. Figure 4 shows the test results of pH ranging from 6.1 to 8.2.

Figure 4.

Experimental results.

While the pH value of the solution is between 6.1 and 7.8, the fluorescence intensity of the quantum dots increases gradually with the increase of the pH value. At pH = 7.8, it reaches a maximum value of 3842. By the end of the rising trend, the quantum dot fluorescence intensity gradually decreases.

The reason is that the stabilizer on the surface of the quantum dot is affected by the change of hydrogen ions in the solution. The quantum dots prepared in paper are in an optimal stable state when the pH is close to 7.8, so the overall fluorescence intensity is the highest when pH decreases. When it increases, the surface stabilizer changes and the defect is weakened, the fluorescence intensity is weakened.

The quantum dot reaches the maximum fluorescence intensity at pH = 7.8. Because the fluorescence intensity of quantum dot is abruptly changed at this time, the test point of pH = 7.8 is removed and a mathematical model is obtained shown in Fig. 5. The mathematical model expression for linear fitting is: Y= 847.68 X – 3582.2.

Figure 5.

pH = 6.1–7.7 linear fitting analysis results.

At this point, the mathematical model R=2 0.9617 and the linearity of the fitting line are smooth.

The linear fitting results of pH = 6.1–7.7 is selected and the obtained model error bars for analysis. The maximum deviation in each point data is 32 and the maximum standard deviation is 83. The slope of the fitted straight line is 847.68 and the measurement sensitivity limit is about 1/847.68 = 0.037.

According to literature research, fluorescence sensitivity of fluorescence spectrometry is about 0.1. So the quantum dot has a higher sensitivity limit and more accurate measurement than organic solvents [13].

According to the experimental process, the quantum dot reaction process can reach the millisecond level. The time required for the whole experimental operation process is around 10 minutes. It can measure the pH of tumor cells quickly. Fluorescence spectroscopy by using an organic solvent needs at least 10 minutes to finish the test. If this operation become more automatically, the test time will continue to be shortened [14].

3.3Temperature impact test results and analysis

The results show that during the temperature rise from 20∘C to 50∘C, the fluorescence intensity of ZnSe quantum dots in PBS buffer at the excitation wavelength of 289 nm is reduced by 37.5% because the thermal activation of surface traps leads to non-radiation of excitons [15]. CdSe quantum dots have similar structure and properties to ZnSe quantum dots and the effect of temperature on their fluorescence characteristics is the same.

Figure 6.

Temperature characteristics at pH values.

After linear fitting, we find that the fluorescence intensity of CdSe quantum dots shows a slight decrease when the temperature increases (in Fig. 6). Temperature coefficient is approximately -17.6 a.u/∘C =-0.02/∘C. According to literature research, in the fluorescence spectroscopy using organic solvents, organic solvents are greatly affected by temperature and have a significant downward trend with temperature increase. The temperature coefficient is -2000 a.u/∘C, far greater than this. The method is less sensitive to temperature and higher efficiency than the fluorescence spectroscopy by using an organic solvent.

4.1Mechanism analysis of pH influence on quantum dots fluorescence intensity

The photoluminescence mechanism of quantum dots is that the low-level electrons of quantum dots are excited by excitation light [16]. The surface modification of quantum dots has a large number of stabilizers. When a physical or chemical reaction causes a change in the surface properties of a quantum dot, the fluorescence or electrochemiluminescence of the quantum dot changes. So the change of hydrogen ion concentration in the solution easily leads to the loss of the surface stabilizer of the quantum dot. The change of the charge of the surface stabilizer leads to surface defects, which causes the quantum yield of the quantum dot to decrease and the fluorescence intensity to decrease. So the pH of liquid to be tested is finally measured.

4.2The possibility of detecting the pH of cancer cells’ metabolic fluid

Many studies have speculated that hypoxia increases the body’s glucose consumption. The adaptation of cells to the acidic home environment is through the use of a potassium exchanger and other proteins, such as carbonic anhydrase, to regulate the pH upregulation. A decrease in pH can result in a variety of protein and structural abnormalities such as DNA helicase and polymerase which plays important roles in DNA replication.

In addition, the acidic microenvironment can promote tumor invasion. As early as 1965, Warburg proposed that tumor cells can undergo strong anaerobic glycolysis to obtain energy. This way produce a large amount of H + and make malignant tumor tissue acidic. In 1984, Wike-Hooley used microelectrode method to determine the pH value of malignant solid tumors which provided a preliminary experimental basis for Warburg’s hypothesis. The most convinced experiment is the application of P magnetic resonance and finds that the extracellular H + of the tumor is higher than that of the cell. The extracellular pH of the malignant tumor is 6–7 and the corresponding normal extracellular pH is 7.1–7.6. The intracellular pH of the malignant tumor is higher than that of the extracellular, but lower than the corresponding normal intracellular pH [17].

The effective test range of the CdSe quantum dots made by the test preparation is 6.1–7.7 and the test range covers the external environment pH of normal cells and tumor cells. The extracellular environmental metabolic fluid is simulated by using PBS buffers of different pH values and finally the CdSe quantum dots are tested for the pH value of the tumor cells.

4.3The possibility of using quantum dot materials to increase the efficiency of pH testing

In the present method, the time for the experiment is not more than 15 minutes (consisted of continuous laser irradiation for 10 minutes and preparation for 5 minutes). However, in practical applications, since the quantum dot fluorescence intensity is stable at the same pH, the actual test does not require continuous laser irradiation for 10 minutes and we can only use one irradiation to obtain the test result. At the same time, because the experimental preparation is currently manual operation, after we mechanize the operation of the method in the future, we can shorten the time for the experiment to 1–2 minutes. Finally, we can measure pH quickly.