A Brief Review Chemiluminescence Sensors Biology Essay

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CL is light produced by a chemical reaction; the energy levels are identical with those involved in fluorescence phenomena, the only difference being the mode of excitation. CL reactions generally yield a product in an electronically excited state producing visible light. As a general rule, only quite exothermic reactions can generate the required energies. Therefore, most CL reactions use oxygen, hydrogen peroxide, or similar potential oxidants. As a principle in CL reactions described in Fig.1, at least two reagents, A and B react to form a product C, some fraction of which is present in an electronically excited state C*, which may subsequently relax to the ground state emitting a photon2:

Or the energy of excited state C* may be transferred to a fluorophor F, leads to produce a exited state F*, which can lately relax to the ground state with emission of a photon3:

Figure 1. Principles of two types of chemiluminescent reactions: (a) direct and (b) sensitized

Hence, the intensity of light emission depends on the rate of the chemical reaction, the efficiency of production of the excited state, the efficiency of light emission from the excited states, and the concentrations of the reactants. CL intensity can be expressed as the following equation4:

is the CL emission intensity (photons/s), is the CL quantum yield, and is the rate of consumption of the initial luminophore (reactant L). The physical significance of is that, under defined experimental conditions, it is the constant of proportionality between and .

Progress in methodology

To my best knowledge, the high-quality CL-based sensors are needed to immobilize CL reagents onto proper substrates. Based on this, the CL sensors would be paid more attention to the following points: (1) the careful selection of CL reagents responding to the analytes, (2) the way to immobilize the reagents, and (3) the substrates selected for reagents immobilization. I’ll step through various immobilization materials in the subsequent section.

3.1 Immobilization of CL reagents on ion-exchange resins

Since most of the established CL systems are operated in aqueous solutions and the CL reagents are various ions, ion exchange resins have been selected as one of the most popular substrates for reagents immobilization. Due to the high surface coverage and convenient immobilization procedure for CL reagents, the ion-exchange approach has been widely used to immobilize CL reagents to develop a series of CL sensors. By injecting some appropriate eluents, the immobilized reagents could be released quantitatively from the ion-exchange column to perform CL reaction. The analytes can also react directly with the immobilized reagents, thus permitting the sensors to operate in a reagent-less way.

The immobilization of luminol and other CL reagents on ion-exchange resins have been extensively employed to develop various CL sensors. Generally, the resins with immobilized CL reagents were mixed and packed into a piece of glass tube, which served as a flow cell and was positioned in front of the detection window of a photomultiplier. The analytes, such as H2O2, could be detected based on the CL reaction of luminol and metal ions bleeding from the ion exchange columns. Problems with this arrangement included unnecessary dilution of the eluted reagents and samples, which reduced the detectability. Also, the need to merge two streams prior to detection made it difficult to miniaturize the configuration.

The consumption of reagents that accompanies CL reactions resulted in the deterioration of the sensors on prolonged use. A promising way to solve this problem was to immobilize CL regents that can recycle. Lin et al.5 have developed a CL sensor by immobilizing tris-(2,2’-bipyridyl)ruthenium(II) complex (Ru(bpy)32+) on the Dowex-50 W cationic ion-exchange resin. Ru(bpy)33+ was electrochemically generated from Ru(bpy)32+ that can be recycled on the electrode. The principle is as follows:

They found that the Ru(bpy)32+ immobilized resin could be used at least for 6 months.

3.2 Immobilization of enzymes on polymer or in sol-gel

The immobilization of enzymes is one of the popular routes to develop chemical and biological sensors, because enzyme acts as high active and high selective catalyst. Highly sensitive and selective CL sensors can be obtained without the consumption of enzyme. Although enzyme-loaded polymer membranes have been widely used to prepare CL sensors, the limited operational stability of the enzyme sensors is the main hindrance to their wider application to solve analytical problems.

The use of sol-gel to immobilize enzymes has become a recognized process for preparing CL sensor. The key advantages of sol-gel are that there is little or no structural alteration of the encapsulate species and it is suitable for optical sensors due to its optical transparency and chemical stability. It has been reported that a CL- H2O2 sensor based on horseradish peroxidase (HRP) immobilized by sol-gel method exhibited excellent characteristics in terms of activity, lifetime and optical transparency6. In another work, Wang et al.7 used glucose oxidase (GOD) and HRP encapsulated in silica sol-gel substrates to develop a novel CL flow-through biosensor for glucose, which further demonstrates the advantages of the sol-gel method. The principle and procedure are described in Fig.2. Glucose oxidase, horseradish peroxidase and luminol were immobilized in one column. The silica sol-gel with glucose oxidase and horseradish peroxidase was immobilized in the first half of the inside surface of a clear quartz tube, and luminol-hybrid Mg-Al-CO3 LDHs were packed in the second half as catalysts and buffer solutions. Compared with conventional luminol CL occurring in the alkaline solution, a stronger CL intensity of the luminol system can emit in weak acid solutions (pH 5.8, while glucose oxidase (GOD) retains its maximum activity at pH 5.5).

Figure 2. Schematic illustration of platform fabricated by bienzyme and luminol-hybrid Mg-Al-CO3 LDHs. Top: illustration of the experimental procedure of the biosensor for glucose.

3.3 Immobilization on other materials

To improve the performance of CL sensors, much attention has been paid to employing new materials as substrates for CL reagent immobilizations.

Molecular imprinting (MI) technique is a rapidly developing technique for the preparation of polymers that would be used as sensing materials to design CL sensors. The imprinted cavities of a defined shape and functional groups in the molecularly imprinted polymer are expected to develop not only with the molecule recognition function but also as a special CL reaction medium. Zhao et al.8 reported a highly selective and high throughput CL-MI sensor for detection of glyphosate (GLY). The procedure is described in Fig.3. Glyphosate-imprinted microspheres were synthesized first and modified on glass sheets, which were placed at the bottom of wells of microplate as the recognizer. After injection of samples with glyphosate, GLY in the solution was selectively adsorbed on the molecularly imprinted microspheres (MIMs) by specific recognition. Several minutes later, the other substances adsorbed by non-specific reaction were washed off with doubly distilled water. At last the CL reagents KMnO4, HCl and Tween-80 were transferred into the well simultaneously by three automatic samplers of Multimode Reader for CL detection.

Figure 3. The schematic representation of CL-MI sensor for detection of glyphosate

The expanding availability of nanoparticles has attracted widespread attention in catalysis due to their high surface areas, high activity and good selectivity. The CL has been detected on the nanosized materials while organic molecules are passing through their surface. In comparison with conventional CL sensors, the sensors based on nanosized materials offer advantages that there is no consumption of CL reagents, and the size of the sensors can be miniaturized due to the small size of nanoparticles. All sensors exhibit good stability and durability. A novel gaseous ester sensor utilizing CL on nano-sized SiO2 has been investigated by Wu et al.9. Ge et al.10 utilized the water-soluble TGA-capped CdTe quantum dots to play a luminophor role in the process of the HClâ€"KMnO4 CL reaction. Their CL sensor was depicted in Fig.4.

Figure 4. CdTe quantum dots and molecularly imprinted polymers modified chemiluminescence sensor in 96 well micro-plate

The immobilization procedure sometimes can be omitted by simply fixing the slight-soluble metal oxides particles onto membranes or in columns. This type of CL sensors offers advantages of simple preparation and long lifetime. For example, the solid-phase manganese dioxide particles have been fixed on the sponge rubber inside the CL flow cell for the detection of analgin with Rhodamine B (RhB) enhancing the CL intensity11. The possible CL mechanism of the reaction may be attributed to the following reactions:

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