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Biosensors and Chemical Sensors for Health Monitoring in the Elderly

May 22, 2020.

 

       Thailand is entering aging society with increasing population older than 60 while the number of working populations is decreasing. This poses an eminent challenge on the development of the country and the well-being of the Thai citizens. One way to create a strong and sustainable aging society is to have a health care system that is easily accessible along with empowering the elderly to be able to perform simple screening for potential health problems themselves. As a result, point-of-care screening systems/devices for early diagnosis become increasingly important. Even though such diagnoses cannot replace thorough investigation by professional healthcare providers in hospitals, they give early warnings to those who are at risk and encourage them to seek proper medical attention before it is too late.  

 

       Biosensors and chemical sensors are interesting technology for developing point-of-care detection system. A biosensor comprises a biorecognition element that is specifically bind to the target analyte and a transducer that converts the binding into a measurable signal. Biosensors can be classified according to the type of the transducer being used, such as electrochemical biosensors and optical biosensors. A chemical sensor is slightly different from a biosensor in such a way that it has a chemical recognition element instead. The advantages of biosensors and chemical sensors when compared with conventional analytical techniques in laboratories are their high specificity and sensitivity, low limit of detection, ease of use and short analysis time. Therefore, biosensors and chemical sensors can potentially be developed into point-of-care screening devices useful for the elderly in the aging society. This research project aims to develop biosensors and chemical sensors to screen microalbumin, creatinine and potassium ions in urine. The three target analytes are potential markers for kidney diseases often found in the elderly.

 

Biosensor for urine microalbumin detection

 

       Microalbumin is the human serum albumin (HSA) that is excreted in urine. A normal microalbumin level does not exceed 373 nanomolar [1] but in patients having kidney damages the concentration is higher. Therefore, microalbumin is considered a key marker for kidney diseases [2]. To develop a biosensor for microalbumin detection, we use an anti-albumin DNA aptamer [3] as a biorecognition element. A DNA aptamer is a single-stranded DNA capable of folding into a specific three-dimensional structure and bind specifically to a target similar to the antigen-antibody binding. Advantages of aptamers over antibodies are ease of syntheses and high stability even in hostile environment [4]. This project utilizes an anti-albumin DNA aptamer in conjunction with the catalyzed hairpin assembly (CHA) technique [5], which is capable of improving the sensor’s sensitivity by producing a large amount of double stranded DNA in the presence of a small amount of microalbumin. In addition, the Förster Resonance Energy Transfer (FRET) technique is used to report the amount of the duplex created by CHA as shown in Figure 1.

 

 

Fig. 1 (A) the sensing mechanism of the microalbumin aptamer based on DNA aptamer in conjunction with (B) the signal amplification method using the catalyzed hairpin assembly (CHA) technique and FRET. The left-hand side of Fig. 1 (C) shows the time-dependent increase in sensor’s fluorescence intensity while CHA is taking place and the right-hand side shows steady state fluorescence intensity increment as the concentration of microalbumin increases.

 

       The binding between microalbumin and DNA aptamer liberates the DNA catalyst (C) (Figure 1A), which then starts the catalyzed hairpin assembly cycle turning the DNA hairpin H1 and H2 into H1:H2 duplex. The cycle continues until the DNA hairpins are depleted. It can be seen that even a small amount of microalbumin can result in a large amount of duplex DNA, which can be easily detected by, for example, labeling a FRET pair on H1 as shown in Figure 1B. The results in Figure 1C (left) show that the fluorescence intensity of the FRET donor (Cy5 in our case) increases with time while CHA cycle is in progress and the rate of fluorescence intensity increment increases with increasing concentration of the DNA catalyst (C). In addition, the fluorescence intensity of the FRET donor increases with increasing microalbumin as hypothesized (Figure 1C, right).

 

Chemical sensor for microalbumin and creatinine detection

 

       Creatinine can be commonly found in mammal’s blood and is produced in muscles from the breakdown of creatine [6]. Creatinine is excreted by kidney filtration and normal level of creatinine in urine is 3.6 - 27.0 millimolar in males and 3.3 - 22.5 millimolar in females [7, 8]. However, the urine creatinine level is lower than the normal levels in patients with kidney damages [9]. Therefore, creatinine is another reliable marker for kidney diseases. The measurement of creatinine level can be done solely or with microalbumin level  (albumin to creatinine ratio or ACR). This research project develops a chemical sensor for the dual detection of microalbumin and creatinine using copper nanocluster as a fluorescence probe. Copper nanocluster is chosen because it is easy to synthesize and cheap. Once functionalized with a suitable ligand, copper nanocluster shows low toxicity, high stability and high fluorescence intensity [10] making it suitable to be used as a fluorescence probe to report the binding between the sensor and the targets of interest. We functionalize copper nanocluster with glutathione (GSH) to improve the stability and to facilitate the interaction between the nanocluster and microalbumin or creatinine under a suitable sensing condition.  Once they interacted, it is expected that the photophysical properties of the nanocluster, such as fluorescence quantum yield, are altered proportionally to the concentration of microalbumin or creatinine making it possible to construct a chemical sensor based on such principle as shown in Figures 2A and 2B.

 

 

Fig. 2 The sensing mechanism of the developed chemical sensor based on copper nanocluster for (A) microalbumin detection and (B) creatinine detection. Figs. 2(C) and 2(D) show fluorescence increment and reduction with increasing concentration of microalbumin and creatine, respectively.

 

       We found that in basic solution the nanoclusters’ fluorescence intensity increases with increasing microalbumin concentration (Figure 2C). This is possibly because in basic solution microalbumin is negatively charged while GSH is positively charged allowing microalbumin to aggregate nanoclusters via electrostatic attraction and there have been reports that copper nanocluster aggregation increases fluorescence intensity [11]. On the other hand, in acidic solution the nanoclusters’ fluorescence intensity decreases with increasing creatinine concentration (Figure 2D). We hypothesize that this is due to the direct interaction between creatinine molecules and copper atoms on the surface of the nanoclusters forming a complex [11] that involve electron transfer between the creatinine and nanocluster, which causes the reduction in the fluorescence emission.

           

Biosensor for potassium ion detection

 

       Potassium ion is very important in maintaining normal cellular functions. It also regulates fluid and electrolyte balances in our body. Prior works suggested that the abnormal levels of potassium ions in body fluids could indicate health problems, such as kidney and primary renal diseases, glomerular nephritis, renal sclerosis and renal failure [12-14].

 

       There are multiple methods for potassium ion detection and using potassium ion-selective electrode, an electrochemical device highly selective for potassium ion, is widely preferred [15]. However, to explore the possibility for constructing another simple analytical methods and portable devices for point-of-care K+ detection, we develop a simple yet sensitive optical biosensor for potassium ion detection based on DNA G-quadruplex (GQ), which is a tertiary structure of DNA comprising layers of four guanine molecules (called G-quartet). GQ requires a cation such as K+ inserted between two adjacent G quartets to screen the electrostatic repulsion between eight oxygen atoms (Figure 3A) [16]. Therefore, it can be seen that the stability of GQ is dependent on the concentration of K+ and one can possibly quantify the concentration of K+ by measuring the concentration of GQ, which can be done readily by, for example, using a GQ specific fluorescent dye that emits bright fluorescence when bound with GQ but show no or low fluorescence otherwise [17]. Thioflavin T (ThT) is selected because it binds GQ with high specificity with dramatic fluorescence enhancement in the bound form. Accordingly, ThT fluorescence intensity should increase with increasing K+ concentration (Figure 3A) and the measured fluorescence emission spectra in Figure 3B support the hypothesis.

 

 

Fig. 3(A) demonstrates sensing mechanism of the developed biosensor for K+ detection based on DNA G-quadruplex and Thioflavin T. Fig. 3(B) shows fluorescence increment with increasing K+ concentration.

 

Study of interactions between Thiflavin T and G-quadruplex DNA by computer simulations

 

       In order to better understand the mechanism of the developed K+ ion sensor, the interactions between Thioflavin T (ThT) and G-quadruplex DNA (GQ) has been investigated with molecular computer simulations. It was found that the twisting of ThT structure upon photo-excitation (photoisomerization process) (Figure 4) competed with the fluorescence emission and thus decreased the fluorescence intensity. From the simulations, there are two modes for ThT and GQ binding that lead to easy and difficult photoisomerization of ThT, as shown in Figure 5A. However, the binding with difficult photoisomerization (inferred from the calculations of photoisomerization free energy as shown in Figure 5B) has more binding stability. This result agrees well with the experiment that the binding of ThT to GQ enhances the fluorescence emission.    

 

 

Fig. 4 Photoisomerization of ThT when its structure undergoes a twisting at the torsion angle j upon photo-excitation from the ground state to the excited state.

 

 

Fig. 5(A) Binding modes between ThT and GQ with difficult and easy photoisomerization (twisting of the structure). Fig 5(B) Photoisomerization free energy of ThT. ThT without binding to GQ (blue line) and ThT binding to GQ with easy twisting (grey line) have low free energy at the twisting angle around 90 degrees. On the other hand, ThT binding to GQ with difficult twisting (green line) has low free energy only at the small twisting angle.

 

Faculty Researchers

 

Assistant Professor Dr. Chittanon Buranachai                   Department of Physics, Faculty of Science,                        

                                                                                            Prince of Songkla University

Associate Professor Dr. Panote Thavarungkul                   Department of Physics, Faculty of Science,                         

                                                                                            Prince of Songkla University

Assistant Professor Dr. Chutintorn Punwong                      Department of Physics, Faculty of Science,                          

                                                                                            Prince of Songkla University

Dr. Sureerat Homhuan                                                        Department of Physics, Faculty of Science,                        

                                                                                            Prince of Songkla University

Dr. Tassaneewan Laksanasopin                                         Biological Engineering, Faculty of Engineering,

                                                                                            King Mongkut's University of Technology Thonburi

Assistant Professor Dr. Chongdee Buranachai                   Department of Chemistry, Faculty of Science,                       

                                                                                            Prince of Songkla University

Associate Professor Dr. Proespichaya Kanatharana          Department of Chemistry, Faculty of Science,                        

                                                                                            Prince of Songkla University

 

Postdoctoral Fellow

 

Dr. Kittirat Phooplub                                                            Department of Physics, Faculty of Science,                       

                                                                                            Prince of Songkla University

 

Students

 

Mr. Wutthinan Thongyod                                                     Department of Physics, Faculty of Science,                       

                                                                                            Prince of Songkla University

Ms. Kwanrudee Chitbankluai                                              Department of Physics, Faculty of Science,                       

                                                                                            Prince of Songkla University

Ms. Supitcha Thammajinno                                                Department of Chemistry, Faculty of Science,                        

                                                                                            Prince of Songkla University

Mr. Anusorn Niammusik                                                      Department of Physics, Faculty of Science,                       

                                                                                            Prince of Songkla University

 

References

 

1.  Wang, R.E., L. Tian, and Y.-H. Chang, A homogeneous fluorescent sensor for human serum albumin. Journal of Pharmaceutical and Biomedical Analysis, 2012. 63: p. 165-169.

2.  Toto, R.D., Microalbuminuria: Definition, Detection, and Clinical Significance. The Journal of Clinical Hypertension, 2004. 6(s11): p. 2-7.

3.  Takenaka, M., et al., DNA-duplex linker for AFM-SELEX of DNA aptamer against human serum albumin. Bioorganic & Medicinal Chemistry Letters, 2017. 27(4): p. 954-957.

4.  Jayasena, S.D., Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics. Clinical Chemistry, 1999. 45(9): p. 1628-1650.

5.  Yin, P., et al., Programming biomolecular self-assembly pathways. Nature, 2008. 451(7176): p. 318-322.

6.  Levey, A.S., R.D. Perrone, and N.E. Madias, Serum creatinine and renal function. Annual review of medicine, 1988. 39(1): p. 465-490.

7.  Ix, J.H., et al., Equations to Estimate Creatinine Excretion Rate: The CKD Epidemiology Collaboration. Clinical Journal of the American Society of Nephrology, 2011. 6(1): p. 184-191.

8.  Boeniger, M.F., L.K. Lowry, and J. Rosenberg, Interpretation of urine results used to assess chemical exposure with emphasis on creatinine adjustments: a review. American Industrial Hygiene Association journal, 1993. 54(10): p. 615-627.

9.  Songjaroen, T., et al., Portable microfluidic system for determination of urinary creatinine. Analytica Chimica Acta, 2009. 647(1): p. 78-83.

10. Zheng, X.-J., et al., One-step, stabilizer-free and green synthesis of Cu nanoclusters as fluorescent probes for sensitive and selective detection of nitrite ions. Sensors and Actuators B: Chemical, 2016. 230: p. 314-319.

11. Shahsavari, S., et al., Ligand functionalized copper nanoclusters for versatile applications in catalysis, sensing, bioimaging, and optoelectronics. Materials Chemistry Frontiers, 2019. 3(11): p. 2326-2356.

12. Modesto, K.M., et al., Safety of Exercise Stress Testing in Patients With Abnormal Concentrations of Serum Potassium††Portions of this manuscript were published in abstract form in Circulation 2002;106(suppl):II-437 (used with permission). The American Journal of Cardiology, 2006. 97(8): p. 1247-1249.

13. Yu, H.-R., et al., Portable Diagnosis Method of Hyperkalemia Using Potassium-Recognizable Poly(N-isopropylacrylamide-co-benzo-15-crown-5-acrylamide) Copolymers. Analytical Chemistry, 2013. 85(13): p. 6477-6484.

14. Hong, Y.H., et al., Twenty-four Hour and Spot Urine Metabolic Evaluations: Correlations Versus Agreements. Urology, 2010. 75(6): p. 1294-1298.

15. Yan, R., et al., Review of progresses on clinical applications of ion selective electrodes for electrolytic ion tests: from conventional ISEs to graphene-based ISEs. Chemical Speciation & Bioavailability, 2016. 28(1-4): p. 72-77.

16. Forman, S.L., et al., Toward Artificial Ion Channels: A Lipophilic G-Quadruplex. Journal of the American Chemical Society, 2000. 122(17): p. 4060-4067.

17. Amdursky, N., Y. Erez, and D. Huppert, Molecular Rotors: What Lies Behind the High Sensitivity of the Thioflavin-T Fluorescent Marker. Accounts of Chemical Research, 2012. 45(9): p. 1548-1557.

 

Reported by

 

Asst. Prof. Dr. Chittanon Buranachai, Department of Physics, Faculty of Science, Prince of Songkla University, A. Hat Yai, Songkhla – 90110, Thailand

E-mail: chittanon.buranachai@gmail.com