Application of Fluorescent lanthanide chelates for bioassay

1. Introduction
Ever since Weissman discovered in 1942 that Eu (III)-beta-diketonato complex absorbs ultraviolet light and gives off visible light, fluorescent chelates of rare earth elements have been researched and developed in various fields.¹⁾ In this article, the application for the measurement of lanthanide fluorescent complexes will be explained.

2. Properties of Rare Earth Elements and their Fluorescent Chelates
Rare earth elements are 15 elements from Lanthane (La) to lutetium (Lu) that are packed with different number of 4f electrons. Their electron structure is indicated as 4f^0-15 5d^0-10 s^1-2. The valence electrons for these elements are 4f electrons, but this orbit is closer to the center than the larger main quantum numbers 5s, 5p, 5d, 6s orbits, and have a special characteristic in that the valence electrons are physically not outer shell electrons, unlike other elements. Since 4f electrons are shielded from the environment from outer layer electrons, they are not easily affected by the surrounding environment. Therefore the properties of rare earth elements closely resemble each other. Due to the characteristics of having this electron structure and an abundance of unpaired electrons, they have been applied widely such as in fluorescent bodies in color televisions, permanent magnets, and laser light emitters. In addition, Gd(III) chelate complex is used as an imaging agent for MRI (magnetic resonance imaging) in the healthcare field. The applications for fluorescent lanthanide chelates include uses as a fluorescent labeling reagents and measuring various materials. Fluorescent rare earth chelates are divided into 3 main groups according to their fluorescent characteristics. In the strong fluorescence group (SM³⁺, Eu³⁺, Tb³⁺, Dy³⁺ chelates) the excitation energy level for the central metal ion is in a slightly lower location than the ligand's excitation triplet (T1) level and can accept the energy transfer from T1. In addition, since there is a large difference between the excitation level and the base level for these ions, non-radiative transition does not occur easily, so the fluorescent quantum yield is high. In the weak fluorescence group (Ce³⁺, Pr³⁺, Nd³⁺, Pm³⁺, Er³⁺, Tm³⁺, Yb³⁺ chelates) the difference between the excitation level and the base level of the central metal ion is fairly small, so the proportion of non-radiative transitioning is large and the fluorescence's quantum yield is low. In the case of Nd³⁺, Er³⁺, and Yb³⁺, chelates that have emissions in the near infrared region have been reported.

Normally, aqueous solutions of Sm³⁺, Eu³⁺, Tb³⁺ and Dy³⁺ produce such a weak fluorescence that it cannot be detected by a regular fluorometer. Once their ions form a chelate with the appropriate ligands, the chlates absorb light from the near-ultraviolet region, become excited, and give off an extremely strong fluorescence. This is because they emit luminescence based on energy transfer to the central metal ion from the ligand of the chelate. La³⁺, Gd³⁺, and Lu3+ chelates do not produce fluorescence (non-fluorescent group).

Excitation and emission occur by the following process. First, the ligand is excited to an excited state (S₁) by the ultraviolet light. Next, by intersystem crossing, energy is transferred to the triplet state T₁, and from that state energy transfer to the europium ion's excited state (⁵D) occurs. Therefore when the metal returns to the base state (⁷F) from the excited state, fluorescence is emitted. At this time, a strong fluorescence emission is observed because b-diketone or other ligand coordinated to the lanthanide ion suppresses the deactivation process that is caused by energy transfer to the solvent molecules.. Accordingly, in order to be a ligand used for prepareing a chelate with a strong fluorescence, it is necessary to have a high absorbance, the energy level of the excited triplet state must be higher than the rare earth ion's lowest excited energy level (⁵D), and energy must be transferred efficiently. Additionally, the intersystem crossing efficiency from the ligand's excited singlet state to the triplet state is greatly affected to the chelate's fluorescence strength. For example, in the case of b- diketone type of ligand indicated in Table 1, the fluorescence of europium is consistently observed at approximately 615 nm regardless of the change in the ligand's absorption maximum wavelength. However, when the absorption maximum wavelength goes over a certain constant value, the chelate stops producing fluorescence. The maximum wavelength relates to the energy level of the excited singlet, and it does not directly indicate the energy level of the excited triplet. However, since there is some correlation between maximum wavelength and energy level of the excited triplet, this event can be explained that the energy level of the excited triplet state is not high enough to transfer to the lowest excited state ⁵D and energy transfer no longer takes place.

In order to understand the properties of fluorescent lanthanide chelates, compare them with organic fluorescent dyes currently used in fluorescence detection. Fluorescent properties of the often-used fluorescent material are indicated in Table 2, and the chemical structures are shown in Fig. 1. In comparison to regular organic fluorescent dye compounds such as Fluorescein and Rhodamine B, the fluorescence of the europium complex has the following four characteristics.






Fig. 1 Examples of fluorescent labels
(a) fluorescein isothiocyanate, (b) rhodamine B isothiocyanate, (c) beta-naphthoyltrifluoroacetone.


(1). The emission wavelength is barely affected by the structure of the ligand.
As stated previously, lanthanide chelates are excited by the ligand's energy absorption, the energy is transferred to the central metal ion due to the energy transfer inside the complex, and fluorescence is emitted when the excited state of the lanthanide ion returns to the base state. In the case of europium chelate, fluorescence based on the transition of 4f-4f is emitted; in most cases ⁵D₀ -> ⁷F₂ emission (approximately 615 nm) is the strongest. That is to say, since the excitation and emission take place in different parts of the complex, lanthanide chelates show the excited spectrum dependent on the ligand's spectroscopic properties, and the emission spectrum which only depends on the central metal ion and not on the ligand. For example, europium complexes always have designative fluorescence spectra as a europium ion complex though there are differences in the fluorescence intensity.

(2). Long fluorescence life span
After energy is transferred from the ligand's excited triplet T₁, the europium chelate produces fluorescence. Since this process contains a relatively slow process compared with the emission of organic fluorescent dyes, the lanthanide chelate has a long fluorescence life span. The 4f orbit on the lanthanide ion is largely shielded by the 5s and 6s orbits, essentially inhibiting transition, which also is a reason for the long fluorescence life. The fluorescence life span of organic fluorescent dye is usually on the nano second level. However, the life span of lanthanide chelates, especially europium and terbium chelates, is greater than hundreds of microseconds. As shown in Table 2, europium fluorescent chelates compared to organic fluorescent dyes have a fluorescent life that's 10⁵ times longer. Using this characteristic, time-resolved fluorescence assay methods have been developed which will be explained in the next section.



(3). The presence of a large Stokes shift
The excitation spectrum and emission spectrum of organic fluorescent dyes overlap at one part, and usually have a mirror symmetric-like relationship. Also, the difference between the excited maximum wavelength and emission maximum wavelength (Stokes shift) is several tens of nm, and in general there is a large overlap in the excited and emission spectrums. On the other hand, in the lanthanide chelate, the ligand is excited by the excitation light, and since fluorescence emission accompanies the lanthanide metal ion's transition to the base level from the excited level after energy is transferred, the Stokes shift is very large, often above 250 nm. Because of this, it does not undergo the concentration quenching (self-quenching) seen in organic fluorescent dyes. More over, when performing fluorescence assay, it has the benefit of being hardly affected by the scattered light derived from excitation light (Fig 2).




Fig. 2 Absorption and fluorescence emission spectra of the fluorescent europium chelate

(4). The emission peak is sharp
The fluorescence emission energy is focused in an extremely narrow wavelength region; the half value of the emission peak is approximately 10 to 20 nm. For example, the fluorescence spectrum of europium at approximately 615 nm is extremely sharp, and the energy of the fluorescence emissions is for the most part concentrated in the wavelength range of 615+10 nm. With such a sharp peak, emission intensity at a specified wavelength is comparatively larger than organic fluorescence dyes that have a broad emission spectrum even if the fluorescence quantum yield is low, and therefore it will be the advantage that can be detected easier (Fig. 2).

3. Advantages of applying lanthanide fluorescence chelates in biochemical analysis.
3-1 Time resolved fluorescence assay method
Conventionally, radioactive isotopes, enzymes, fluorescent compounds, chemiluminescent compounds, and various probes (including labeling agents) have been used in biological sample analysis. In particular, due to the ease of handling and high sensitivity of detection of fluorescent probes, they have been used and played a very important role in base sequence analysis of genes and expression analysis with a DNA micro array. Methods that employ fluorescent probes have the advantages such as no isotope requirement, easy handling, a wide range of applications due to the abundance of the probes, simple detection, and high sensitivity. In the past, the organic fluorescent dyes such as Fluorescein and Rhodamine have been used. However, in bioassays using organic fluorescent labeling agents, the background fluorescence derived from scattered excitation light and emissions of coexisting material in the sample largely inhibits the detection of the emission signal from the labeling agent, thus making it difficult to get a highly sensitivity measurement is a disadvantage.

Differing from the fluorescence of organic compounds, the fluorescence of lanthanide fluorescent chelates as stated in the previous paragraph have the following characteristics: (1) long fluorescence life span, (2) a large Stokes shift, and (3) a sharp emission peak. Fluorescence assays that employ lanthanide fluorescent chelates use a time-resolved fluorescence assay differing from conventional fluorescence assay methods. Time-resolved fluorescence assay is a method that utilizes the difference in the fluorescence life of material to selectively test for targeted compounds that produce fluorescence with a long life span. The principle behind the use of lanthanide fluorescent chelates in time-resolved measurement is shown in Fig. 3. When excitation light is irradiated with pulse, the coexisting impurities and the material of the container along with the target compound are excited and produce emission. At this point, the scattered light from the excitation light is also detected. However, the fluorescence from the coexisting impurities and the material of the container fades quickly because of their short fluorescent life span. On the other hand, since the fluorescent life span of the lanthanide fluorescent chelates is more than several hundred micro seconds, it is possible to detect the fluorescence of the target compound with good sensitivity after the background fluorescence from the impurities have faded sufficiently. In short, time-resolved fluorescence assay methods that employ lanthanide fluorescent chelates take advantage of the chelate's special properties, skillfully using the difference between the fluorescence life span of the fluorescence labeling agent and background fluorescence, and effectively limiting the background fluorescence from the impurities in the sample and the measurement equipment, making it possible to selectively detect only the fluorescence with a long life span emitted from the probe. Thus the time-resolved fluorescence assay accomplished a highly sensitive and accurate measurement that in the past has not been available.



Fig. 3 Measurement principle of time-resolved fluorometry

Since the 1980's, according to the fluorescent property of the lanthanide fluorescent complex, various measurement methods for biologically related materials, such as time-resolved fluorometric immuno assay, DNA hybridization assay, cell activity assay, fluorescent bioimaging, HPLC and so on, have one after another been developed by using lanthanide fluorescent chelates as a probe, and the range of those applications is expanding. Using immunoassay as an example, the applications for the analysis of lanthanide fluorescent chelates will be addressed in this article.

3-2 Using lanthanide chelate probes in biological component assays
In bioassays, various probes (including labeling agents) such as radioactive isotopes and enzymes, fluorescent compounds, and chemiluminescent compounds have been frequently used. In particular, fluorescent probes have been used and play a big role in the human genome project and biochips because of the ease of detection and high sensitivity. Methods that employ fluorescent probes have the following advantages: no radioactivity issue, easy to handle, a wide range of possible applications due to the variety of types, easy to detect, high sensitivity, and so on. In the past, organic fluorescent compounds such as Fluorescein and Rhodamine have been commonly used. However, bioassays that employ organic fluorescent labeling agents have a disadvantage in that fluorescent detection is greatly inhibited by back ground noise derived from scattered excitation light and fluorescence from coexisting material that's present in the sample, making it difficult to obtain a highly sensitive measurement. Applications for biological analysis of lanthanide fluorescent chelates will be addressed, including the view as a means of solving the problems of these types of organic fluorescent labeling agent. In addition, due to a limit of the number of references that can be listed, each source was not stated individually. Please check reviews of these cases.^2-10)

3-3 Dissociation Enhanced Lanthanide Fluoroimmunoassay (DELFIA method)
This method employs the Eu³⁺ chelate of N¹(p-isothiocyanatebenzyl)-ethylenediamine tetraacetic acid (SCN-Ph-EDTA-Eu³⁺) or the Eu³⁺ chelate N¹-(p-isothiocyanatebenzyl)diethylene triamine-N¹,N²,N³,N³-tetraacetic acid (SCN-Ph-DTTA-Eu³⁺) as a labeling agent, and analysis is conducted by the principles shown in Fig. 4. In this method, europium chelate labeling protein (antibody or an antigen) is prepared first. After an immunoreaction involving these labeling proteins, unreacted reagent and immunocomplex are separated (B/F separation). beta-diketone (in most cases, 2-naphthoyltrifluoroacetone beta-NTA) is added on the obtained immunocomplex, and then weak acidic fluorescence enhancement solution (micelle solution, pH=3.2) contains trioctylphosphineoxide (TOPO) and the detergent, Tritonx-100, is added. Eu³⁺ ion from the europium labeling agent in the immunocomplex is combined with the ligand in the solution, the solution is transformed into the strong fluorescent micelle solution of Eu(b-NTA)₃(TOPO)₂ chelate, and this solution is applied for time resolved fluorescence assay.

In this method, both labeling agents SCN-Ph-EDTA-Eu³⁺ and SCN-Ph-DTTA-Eu³⁺ themselves are non-fluorescent chelates, but by converting the europium in the labeling agent to the fluorescent complex, Eu(beta-NTA)₃(TOPO)₂ (10^-14 M-level detection limit), which is capable of a highly sensitive detection, it is possible to measure its fluorescence. However, in fluorescence assays, it is necessary to add what is called fluorescence enhancement solution, which contains beta-NTA-TOPO-Triton X-100. In this fluorescence enhancement solution, there is a large excess of ligand (b-NTA and TOPO), so when Eu³⁺ enters from the outside of the system, it is concerned that a strong background fluorescence is emitted. Accordingly, this method has the disadvantage of being easily affected by europium contamination.



Fig. 4 Assay principle of DELFIA system

3-4 Time-resolved fluorescence immunoassay method
In this method, first lanthanide chelate labeling protein (antibody or antigen)is prepared. An immune response using this labeling protein is carried out, then time resolved fluorescence assay is conducted while in a solid phase after the unreacted reagent and immune complex have been separated. One of the assays of this method is known as FIAgen time-resolved fluoresent immunoassay which employs fluorescent europium chelate, 4,7- bis(chlorosulfophenyl)-1,10- phenantroline-2,9-dicarboxylate Eu³⁺ complex (BCPDA-Eu³⁺), as a labeling agent. Compared to the DELFIA method, the advantages of the FIAgen time-resolved fluoresent immunoassay are that no fluorescence enhancement solution is required, and no europium contamination from the buffer solution and the assay environment is anticipated. However, there is a disadvantage that the sensitivity is low because the fluorescence of the labeling agent BCPDA-Eu³⁺ which is used in this method is weak (detection limit of 10^-11 M).

Next, a ligand which was developed in this laboratory, chlorosulfonyl beta-diketone compound, 4,4'-Bis (1",1",1",2",2",3",3",-heptafluoro-4",6",-hexanedion-6"-il)chlorosulfo-o-terphenyl (BHHCT, Fig 5), will be introduced. This compound reacts with proteins that have an amino group via chlorosulfonyl group, and the protein is labeled by generating a sulfonamide bond (-SO₂-NH-). When an adequate amount of EuCl₃ is added to the labeled protein solution, it quickly becomes BHHCT-Eu³⁺ fluorescent-labeled protein. In recent years, high sensitivity time-resolved fluoresent immunoassays using BHHCT-Eu³⁺ as a labeling agent have continuously been developed and applied in the measurement of various materials. For the most part, BHHCT-Eu³⁺ labeled streptavidin (SA), SA-BSA (bovine serum albumin) conjugate, antibody, and hapten-BSA conjugate are employed in these measurements. Because BHHCT-Eu³⁺ has an extremely strong and very long fluorescent life span, time-resolved fluoresent immunoassays that employ this labeling agent do not require fluorescence amplification solution, and a solid phase time resolved fluorescent assay can be performed once the reaction is finished.



Fig. 5 Structure of BHHCT-Eu³⁺

There are several examples in which BHHCT-Eu³⁺ labeling agent is used in a non-competitive (sandwich) time-resolved fluorescent immunoassay, such as AFP, IgE, thyroid stimulation hormone(TSH), Stromal-cell derived factor-1 (SDF-1), and cytokine type protein (interleukin-1 aplha, tumor necrosis factor alpha and interpherone gamma) from human blood serum, and examples in a competitive time-resolved fluorescent immunoassays, such as the measurements of the stimulant drug metamphetamine in human urine and hair, p21 protein in human blood serum, the pesticide bensurufuron-methyl in environmental water, estardiol, and estriol.^{5, 10)}

3-5 Oxygen amplification time-resolved fluorescent immunoassay
EDTA-Tb³⁺ complex is a non-fluorescent complex, but when it generates a ternary complex with salicylic acid derivative, Tb³⁺ produces fluorescence with a particularly long life span. Utilizing this reaction, an enzyme amplification time-resolved immunoassay method was developed in which an enzyme alkaline phosphatase (ALP) and phosphoric acid ester of 5-fluorosalicylic acid were used as a labeling agent and a substrate, respectively.¹¹⁾ As shown in figure 6, after an immunoreaction is performed using ALP labeled antibody, 5-fluorosalicylic acid phosphoric acid ester solution (pH = 9-10) is added. After the substrate is hydrolyzed by ALP's catalytic action and becomes 5-fluorosalicylic acid, EDTA-Tb³⁺ (pH=13) solution is added, generating a 5-fluorosalicylate-Tb³⁺-EDTA ternary fluorescent complex to be used in time-resolved fluorescence assay. Diflunisal phosphoric acid ester also can be used as an enzyme substrate instead of 5-fluorosalicylic acid phosphoric acid ester. In this case, the chelate used in time-resolved fluorescence assay is the EDTA-Tb³⁺ and diflunisal ternary fluorescence complex. Alpha-fetoprotein (AFP) and prostate specific antigen (PSA) in human blood serum have been have been reported as examples of a biological sample analysis which uses this method.¹²⁾



Fig. 6 Detection principle of enzyme-linked time-resolved immunoassay

3-6 Homogeneous time-resolved fluorescent immunoassay
The advantages of homogeneous immunoassay are no requirement of a solid phase material, no separation of the bound form from the free form (B/F separation) steps, no washing steps, and a fast measurement can be expected. A homogeneous time-resolved fluoresent immunoassay that utilizes the long life fluorescence properties and the movement of fluorescence resonance energy transfer between one fluorescent compound to another has been developed. A representative example is the homogenous time-resolved fluorescent immunoassay called time-resolved amplified cryptate emission (TRACE). In the TRACE method, first, 2 types of antibodies that can react at the same time with one antigen are each labeled with the two fluorescent labeling agents trisbipyridine cryptate-Eu³⁺ (fluorescent energy transition donor dye, abbreviated as TBP-Eu³⁺) and allophycocyanine (fluorescent energy transfer acceptor dye, cross-linked allophycocyanine, a dye protein with a molecular weight of approximately 104 kD, 665 nm fluorescence maximum wavelength, approximately 0.7 fluorescence quantum yield, abbreviated as XL665). The two types of labeled antibodies join with the antigen, and the donor dye and the acceptor dye come close together. When the donor dye is irradiated at its excited wavelength, the donor dye's fluorescence emission energy moves to the acceptor dye, and the fluorescent energy derived from the transfer makes the acceptor dye produces a particular fluorescence signal. Since the donor dye's fluorescence emission life span is extremely long (approximately 1 ms), the acceptor dye's fluorescence emission from the fluorescence energy transfer also has a long fluorescence life. Accordingly, the long-lived fluorescence of the acceptor dye can now be analyzed using time-resolved fluorescence measurement. The unreacted XL665 labeled antibody in the solution does not produce an emission by ultraviolet excitation, so the emission from the free XL665 is eliminated. Due to the extremely sharp fluorescence emission peak of lanthanide chelates (possessing long fluorescence life), overlapping of lanthanide chelates emission in the measured wavelength of XL665 is minimal and there is little interference with the measurement. Actually, when measuring biological samples utilizing this method, fluorescence intensity ratio between XL665 at 665nm and TBP-Eu³⁺ at 620nm is used as a signal. This is because to adjust the effects due to the heterogeneity of photo absorption and quenching in the biological sample and between sample to sample. Figure 7 indicates the structure of TBP-Eu³⁺ and the principle of the TRACE method. There are several assays that have been reported such as detection of AFP and PSA in human blood serum, human immunodeficiency virus protease assay, tyrosine protein kinase assay, and p53/HDM2 protein-protein binding assay.¹³⁾



Fig. 7 tructure of TBP-Eu³⁺ and principle of "TRACE"-homogeneous time-resolved fluoroimmunoassay

The small molecule organic fluorescent dyes such as rhodamine, Cy3, and Cy5 have been reported as fluorescence energy transfer receptor dyes in homogenous time-resolved fluorescent immuno assay other than XL665. Terbium fluorescent chelate must be employed as a donor pigment when using Cy3 and Rhodamine, europium fluorescent chelate must be employed when using Cy5, in order to match the absorption wavelength and the fluorescent emission wavelength to the lanthanide fluorescent chelate used as the donor dye for the energy transfer. These methods have been already applied in the measurements of free beta-subunit of human chorionic gonadotrophin, bensulfuron-methyl, and interaction detection between human interleukin-2 and interleukin-2 receptor.

4. Multi-color measurement
Trivalent europium, samarium, terbium, and dysprosium lanthanide fluorescent chelates have different fluorescent wavelengths and sharp fluorescence peaks, so by combining these chelates as labeling agents, it is possible to analyze multiple components, namely simultaneous multiple components detection is possible.

Since the sensitivity of samarium is usually not as high as that of europium, in most cases Eu-Sm dual-label time-resolved fluorescent immunoassay is used for simultaneous measurement of two components, the low concentration component (using europorium labeling) and high concentration component (using samarium labeling) in samples. Examples of this simultaneous assay include lutropin in human blood serum and follitropin,¹⁴⁾ myoglobin and carbonate dehydratase, AFP and the free b-subunit of human chorionic gonadotropin, pregnancy serum protein A and free b-subunit of human chorionic gonadotropin as well as AFP in a serum from a pregnant woman, human chorionic gonadotropin and estriol. Simultaneous detection of free PSA in human blood serum and whole PSA is reported as an example of Eu-Tb dual-label time-resolved fluorescent immunoassay.¹⁵⁾

It is also possible to simultaneously measure two components in a single sample by Eu-Sm dual-label time-resolved fluorescent immunoassay by the combination of BHHCT-Eu³⁺ and BHHCT-Sm³⁺ which was developed in our laboratory. Simultaneous measurements of AFP and CEA (carcinoembryonic antigen) in human blood as well as simultaneous measurements of two types of drugs are examples of that application.

In addition, a high sensitivity simultaneous measurement of AFP in human blood serum and CEA by Eu-Tb dual-label time-resolved fluorescent immunoassay is also being developed using BHHCT-Eu³⁺ combined with terbium fluorescent labeling reagent BPTA-Tb³⁺, which has a strong fluorescence and a long fluorescence life, instead of using BHHCT-Sm³⁺. This method as shown in Fig. 8 takes following steps: First, the 96-well plate is coated with anti-AFP antibody and anti-CEA antibody mixture. After the reaction with human blood serum, the reaction with BHHCT-Eu³⁺ labeled anti-AFP antibody and biotin-labeled anti-CEA antibody mixture is followed on the plate. When the reaction has finished, the plate is washed, the florescence intensity of BHHCT-Eu³⁺ is measured at 615 nm, and then the concentration of AFP is calculated. In continuation, BPTA-Tb³⁺-labeled SA is added, and after the reaction with biotinylated anti-CEA antibody, the plate is washed, the fluorescence intensity of BPTA-Tb³⁺ is measured at 545 nm, then the concentration of CEA is calculated.



Fig. 8 Dual-label time-resolved fluoroimmunoassay of AFP and CEA with fluorescent lanthanide labels, BHHCT-EU³⁺ and BPTA-Tb³⁺

In this way, the reason simultaneous measurement of highly sensitive AFP-CEA without fluorescence enhancement solution is possible (detection limit: AFP, 44 pg/ml; CEA, 76 pg/ml) is due to the use of strong fluorescent labeling agent Eu-Tb.

In Eu-Sm dual-label time-resolved fluorescent immunoassay, it is possible to use SCN-Ph-DTTA-Eu³⁺ and SCN-Ph-DTTA-Sm³⁺ as a labeling agent, and it is possible to use DELFIA fluorescence enhancement solutions that includes b-NTA, TOPO, and Triton X-100 without modification. Since Tb3+ and Dy³⁺ in this fluorescence enhancement solution are non-fluorescent, this enhancement solution was employed for the 4 component simultaneous measurements of TSH, 17 a-hydroxyprogesterone, immunoreactive trypsin, and creatine kinase MM isoenzyme in human blood serum by Eu-Sm-Tb-Dy quadruple-label time-resolved fluorescent immunoassay. The solution included pivaloyltrifluroacetone, Y³⁺, Triton X-100 and 1,10-phenanthroline.¹⁶⁾ In this fluorescence enhancement solution, Eu³⁺, Sm³⁺, Tb³⁺, and Dy³⁺ ions produce chelates with a fluorescent life span of 820, 88, 323, 27 ms, respectively. The detection limits of each ion are 0.035(Eu³⁺), 7.9(Sm³⁺), 0.34(Tb³⁺), 46(Dy³⁺) pM.

5. Method for high sensitivity
In the previous sections, lanthanide fluorescent chelates as labeling agents in high sensitivity analysis were explained from a qualitative perspective (by their properties). In this section, an approach to high sensitization from a quantitative perspective will be introduced. In this approach, the lanthanide fluorescent chelate does not directly label the antibody, but rather after forming covalent bonds with polymer macromolecules or being embedded by macromolecules, the macromolecule forms a compound with the antibody and is employed in analysis. Following 3 methods have been reported:

(1) The method that uses BCPDA-Eu³⁺ labeled polyvinylamine-biotin-streptoavidin complex:^17-20) In this method, first biotin-labeled polyvinylamine (biotin)_x-PVA is prepared, then BCPDA is used to label (biotin)_x-PVA. When the obtained (biotin)_x-PVA-(BCPDA)_y (x=5-10; y=50-100) is mixed with a certain amount of streptavidin (SA) and Eu³⁺ solution, the macromolecule (SA)_z-(biotin)_x-PVA-(BCPDA-Eu³⁺)_y is obtained and can be used directly in time-resolved fluorescent immunoassay. This complex is used in analysis.

(2) The method that involves europium fluorescent chelate labeled poly(Glu:Lys)- streptavidin complex and europium fluorescent chelate labeled poly(Glu:Lys)-BSA streptavidin complex:²¹⁾ In this method, first poly(Glu:Lys) is labeled with Eu³⁺ chelate prepared by 4-[2-(4-isothiocyanatophenyl)ethynyl]-2,6-bis{[N,N-bis(carboxymethyl)amino]methyl}pyridine}. When the prepared labeled poly(Glu:Lys) is combined with streptavidin, europium chelate labeled poly(Glu:Lys)- streptoavidin complex is obtained. After poly(Glu:Lys) is combined with BSA and then with streptavidin, the resultant is europium chelate-labeled poly(Glu:Lys)-BSA-streptavidin complex. Two types of fluorescent macromolecular complexes are applied in the high sensitivity time-resolved fluorescent immunoassay for human PSA, and each has shown to have a detection limit of 4 pg/ml and 6 pg/ml respectively.

(3)The method that utilizes polystyrene latex nanoparticles including europium fluorescent chelate:^22-26) In this method, first a 107 nm diameter polystyrene nanoparticle that includes over 30000 molecules of beta-diketone-Eu³⁺ fluorescent complex is used as a fluorescent probe to label streptavidin and antibody. Labeled streptavidin and antibody are used in time-resolved fluorescent immunoassay. Time-resolved fluoresent immunoassay for PSA that uses this method has a detection limit of 0.21 pg/ml.

6. Closing
The uses of lanthanide fluorescent chelates as labeling agents were introduced in this article. Currently in biochemical analysis, organic fluorescent dyes and enzymes (such as alkaline phosphatase and peroxidase) are widely used as labeling agents. Including the lanthanide chelates presented in this article, all labeling agents have their advantages and disadvantages, so it is wise to choose the labeling agent that will make maximum use of those advantages corresponding to the desired analysis. Though labeling by lanthanide chelates is not widely in use, the biggest merit is that analysis can be performed with even crude material because time-resolved analysis can be employed due to the long fluorescent life span. In recent years, study examples of proteomics has been on the rise, and is expected to play a big part fields where the subject of analysis is other than materials that can amplify PCR of DNA in vitro. In this sense, this article comes to a close with the continued hope that lanthanide chelates will gain recognition.

References
1) S. Weissman, J. Chem. Phys., 10, 214 (1942).
2) E. Soini and I. Hemmila, Clin. Chem., 25, 353-361 (1979).
3) I. Hemmila, Appl. Fluoresc. Technol., 1, 1-8 (1988).
4) I. Hemmila, Scand. J. Clin. Lab. Invest., 48, 389-400 (1988).
5) J. Yuan and K. Matsumoto, Bunsaeki, 1998, 873-880.
6) J. Yuan and K. Matsumoto, Bunseki Kagaku, 48, 1077-1083 (1999).
7) I. Hemmila and V.-M. Mukkala, Crit. Rev. Cl. Lab. Sci., 38, 441-519 (2001).
8) K. Matsumoto and J. Yuan, in Metal Ions in Biological Systems, Vol. 40, ed. A. Sigel and H. Sigel, Marcel Dekker, Inc., pp. 191-232 (2003).
9) J. Yuan, G. Wang and K. Matsumoto, Trends Inorg. Chem., 7, 109-117 (2001).
10) J. Yuan and K. Matsumoto, Tanpaku Kakusan Kouso, 48, 1550-1558 (2003).
11) R. A. Evangelista, A. Pollak and E. F. G. Templeton, Anal. Biochem., 197, 213-224 (1991).
12) H. Yu, E. P. Diamandis, A. F. Prestigiacomo and T. A. Stamey, Clin. Chem., 41, 430-434 (1995).
13) S. A. Kane, C. A. Fleener, Y. S. Zhang, L. J. Davis, A. L. Musselman and P. S. Huang, Anal. Biochem., 278, 29-38 (2000).
14) I. Hemmila S. Holttinen, K. Pettersson and T. Lovgren, Clin. Chem., 32, 2281-2283 (1987).
15) S. Eriksson, M. Vehniainen, T. Jansen, V. Meretoja, P. Saviranta, K. Pettersson and T. Lovgren, Clin. Chem., 46, 658-663 (2000).
16) Y.-Y. Xu, K. Pettersson, K. Blomberg, I. Hemmila H. Mikola and T. Lovgren, Clin. Chem., 38, 2038-2043 (1992).
17) A. Scorilas and E. P. Diamandis, Clin. Biochem., 33, 345-350 (2000).
18) A. Scorilas, A. Bjartell, H. Lilja, C. Moller and E. P. Diamandis, Clin. Chem., 46, 1450-1455 (2000).
19) L.-Y. Luo and E. P. Diamandis, Luminesc., 15, 409-413 (2001).
20) A. Scorilas, A. Magklara, B. R. Hoffman, R. M. Bromberg, A. Bjartell and E. P. Diamandis, Anal. Sci., 17 Suppl., i547-i550 (2001).
21) Q.-P. Qin, T. Lovgren and K. Pettersson, Anal. Chem., 73, 1521-1529 (2001).
22) H. HarmaT. Soukka, S. Lonnberg, J. Paukkunen, P. Tarkkinen and T. Lovgren, Luminesc., 15, 351-355 (2000).
23) H. HarmaT. Soukka and T. Lovgren, Clin. Chem., 47, 561-568 (2001).
24) T. Soukka, H. HarmaJ. Paukkunen and T. Lovgren, Anal. Chem., 73, 2254-2260 (2001).
25) T. Soukka, J. Paukkunen, H. HarmaS. Lonberg, H. Lindroos and T. Lovgren, Clin. Chem., 47, 1269-1278 (2001).
26) T. Soukka, K. Antonen, H. Harma A.-M. Pelkkikangas, P. Huhtinen and T. Lovgren, Clin. Chim. Acta, 328, 45-58 (2003).