Studies On The Chemiluminescence Reaction Of Guanine And Its Analytical Application

Guanine can specifically react with TMPG to emit instantaneous chemiluminescence(CL). This CL system is simple and rapid, and can recognize nucleid acids directly. Thus, it has great advantages in biosensing for DNA recogniztion. Our research mainly focused on this CL system and established some new methods for enzyme and ion detection.1. Study of guanine CL systemIn this work, we studied the guanine CL system and explored the main factors which may influence the CL intensity. We also explored the influence on CL intensity with the different sequence of DNA and different configuration of DNA. Meanwhile, we selected the best reaction conditions and explored chemical resonance energy transfer(CRET) between guanine chemiluminescence and fluorescent molecules. Our research laid a foundation for the application of guanine chemiluminescence.2. Simple detection of Exonuclease I activity based on chemiluminescence resonance energy transferIn this work, we designed a probe PI (5'-biotin-AAA AAA AAA AGA GAG AGA GAG AGA GAG AG-FAM-3') with 5'terminus end labeling biotin and 3'terminus end labeling FAM. In the absence of Exo I, the strong force between biotin and streptavidin connected probe PI with magnetic beads(MBs) labeled with streptavidin. With the addition of TMPG, CRET happened to emit strong chemiluminescence. In the presence of Exo I, probe PI underwent the degradation process from 3'terminus end. As a result, the final G-rich DNA sequence attached to MBs became shorter and no CRET happened between TMPG-guanine-FAM, so the CL intensity decreased largely. We managed to detect the activity of Exo I according to the change of CL intensity, and the detection limit was 1.1 x 10-5 U/jxL. In conclusion, we applied guanine chemiluminescence into Exo I detection, and this method was expected to be used for detection of Exo I in complex samples.3. Rapid and sensitive detection of potassium ion with guanine chemiluminescenceA simple and rapid method for detection of potassium ion (K+) was presented based on guanine chemiluminescence (CL) system. In this system, one G-rich DNA molecular was used as the recognition element. K+ can cause the G-rich DNA to form G-quadruplex conformation, resulting in remarkable quenching of guanine CL intensity of G-rich DNA. The CL intensity of this CL system decreased with increasing K+ concentration, to reveal a linear relationship in K+ concentration range from 3×10-5 M to 1×10-3 M. One whole detection process can be finished in about 5 min. Other common cations (such as Na+, NH4+, Mg2+, Ca2+, Zn2+and Pb2+) did not cause notable interference on the K+ detection. The mechanism of this strategy was also discussed. The sensing strategy was low-cost and simple without requirement for complex labeling of probe DNA. This reported scheme can applicable to the detection of other G-rich aptamer-binding chemicals or biomolecules.4. Guanine chemiluminescence for uracil-DNA glycosylase (UDG) detection through chemiluminescence resonance energy transferAs was found, the CL intensity of G-rich single stranded DNA was almost the same as G-rich double stranded DNA. Whereas,the CL intensity of G-rich DNA was different from that of G-quadruplex. Based on this phenomenon, we established a new mehod for UDG activity detection with CRET between TMPG-guanine-FAM. We designed a double stranded DNA probe. One part was G-rich DNA with U base, and the other part was its complementary sequence. In the absence of UDG, double stranded DNA and TMPG can emit strong CL with CRET. In the presence of UDG, the U site would be hydrolysed, resulting in the dissociation of ds-DNA. In the presence of K+, G-rich single DNA converted into G-quadruplex to give low CL intensity. We managed to detect the activity of UDG with this phenomenon. Our method was quite simple and rapid with the detection limit was 0.3 U/mL. Meanwhile, we also applied this method to detect the inhibition of UGI and detection of UDG in Hela cells.