Chem. Listy 91, 477 - 482 (1997)

LUMINESCENT TECHNIQUES APPLIED TO BIOANALYSIS

STEFANO GIROTTIa, ELIDA NORA FERRIa,SEVERINO GHINIa, FABIÁNA FINI3, MONICAMUSIANIb, GIACOMO CARREAC, ALDO RODAd,PAVEL RAUCHe

"institute of Chemical Sciences, University of Bologna,Institute ofMicrobiology, University of Bologna, c Institute

of Hormone Chemistry, CNR, Milana, Department ofPharmaceutical Sciences, University of Bologna, Italy, In-stitute of Chemical Technology, 166 28 Prague

Received January 21, 1997

Content

1. Introduction2. Luminescent systems

2.1. Firefly bioluminescence2.2. Bacterial bioluminescence2.3. Luminol chemiluminescence2.4. Dioxetane chemiluminescence2.5. Acridans chemiluminescence

3. Immobilized flow systems3.1. Apparatus3.2. Applications3.3. Microdialysis technique

4. DNA and RNA probes5. Conclusion

1. Introduction

The bio- and chemiluminescent reagents, introduced inanalytical chemistry since the seventies, allow amplification ofthe analytical signál and to develop fast and sensitive methods1.The bioluminescent enzymatic systems based on firefly orbacterial luciferase, the chemiluminescent emission of thehydrogen peroxide/peroxidase/luminol systém or some morerecently introduced luminescent substrates (dioxetanes) of-fer a unique and universal tool to determine the majority ofanalytes and enzymes of potential interest in bioassays2.

It has been demonstrated that the best performanceswere obtained using the immobilized enzymes, because ofan increased stability, catalytic activity, the possibility torecover the enzymes and to develop automated systems.Immobilized enzymes are widely ušed in the biochemicallaboratory to assay several analytes and enzyme activities.Metabolite analysis in biological systems requires sensitive andspecific methods because analytes are present at low con-centrations and further dilution of biological samples isoften needed to avoid interference due to matrix effects. Inconjunction with bioluminescent enzymes (firefly and bac-terial luciferases) and chemiluminescent catalysts (peroxi-dase and alkaline phosphatase)-^, we set up high-sensitiveflow sensors based on the use of nylon tube coil or epoxymethacrylate (Eupergit C) column as solid support. When theseflow sensors were applied to in vivo determination, a suitablemicrodialysis probe inserted directly into tissues or bloodallows continuous measurement of extracellular analyte levelsby means of a bioluminescent flow detector systém.

In the diagnosis of several microbial diseases, the useof nucleic acid hybridization techniques has led to the useof several radioactive or non isotopic labelled probes whichcan be immunoenzymatically visualized7. It has also beendemonstrated that chemiluminescent enzyme substrates arethe most sensitive tools available for the detection of en-zyme conjugates, including the nucleic acid hybridizedprobe8. The hybridized DNA and RNA probes are gener-ally visualized by Fab fragments labelled with alkalinephosphatase (ALP) or horseradish peroxidase (HRP)9.

Here we summarize our experience with immobilizedluminescent enzymes coupled with the analyte specificenzyme and DNA and RNA chemiluminescent probesapplied to viral detection.

2. Luminescent Systems

2 . 1 . F i r e f l y B i o l u m i n e s c e n c e

Firefly luminescence is the most extensively studiedbioluminescent systém4-10. The light-producing reactionrequires the enzyme luciferase, luciferin, Mg2+, ATP, and

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molecular oxygen, which give an oxyluciferyl adenylate-enzyme complex in the excited statě. After emission (560nm), the ground-state complex dissociates to form enzyme,AMP, pyrophosphate (PPi), oxyluciferin, and carbon dioxide:

ATP + luciferin + O2 l u C l f e r a S e ) AMP + PPi + CO2 +

+ oxyluciferin + LIGHT

The concentration of other metabolites, of cofactorsinvolved in the luminescent reaction like Mg2+, or theactivity of enzymes participating in coupled reactions lead-ing to the formation or consumption of ATP can beassayed with the aid of the firefly luciferase-dependentreaction1'3.

2 . 2 . B ac t er i al B i o 1 um i n es c e n ce

The bioluminescent enzyme systém from marine bacte-ria1"4 consists of a NAD(P)H:FMN oxidoreductase anda luciferase which emits light at 490 nm in the presence ofFMN, NAD(P)H, a long chain aliphatic aldehyde and mo-lecular oxygen, according to reactions (7) and (2).

Various substances of biological interest and enzymeactivities can be analysed by coupling the luciferase and theoxidoreductase to a third reaction which either produces orconsumes NADH or NADPH.

2 . 3 . L u m i n o l C h em i 1 u m i n e s ce n c e

The amount of H2O2 which derives from the catalysisof a specific oxidase may be assayed by adding excessperoxidase and luminol2"4 and measuring steady statě che-miluminescence intensity according to the scheme:

luminol + 2 H2O2 + OH" — > aminophthalate +

+ 3 H2O + N 2 + LIGHT

Since the quantum yield of this reaction is very low(-10 %), the addition of enhancers, such as p-iodophenolor/7-hydroxycinnamic acid, in the systém further increaseslight emission and transforms the signál from a flash toa steady statě output1!. The enhancers also reduce the back-ground light emission from the luminol-oxidant mixture,further improving the signal/background ratio.

Another field which luminescent detection systems cancontribute significantly to the analytical performance ofvarious assays is that of immunoassays, in which the highsensitivity of luminescent substrates can be coupled to thespecificity of immunological reagents such as polyclonalor monoclonal antibodies12.

2 . 4 . D i o x e t a n e C h e m i 1 u m i n e s c e n c e

New alkaline phosphatase chemiluminescent substratesare the adamantyl-1,2-dioxetane phenyl phosphate (PPD)and its Cl- and Br- derivatives. In the presence of alkalinephosphatase, PPD is dephosphorylated and produces anunstable intermediate which further decomposes, emittinga glow of light in direct proportion to the amount of alkalinephosphatase6:

[F]*->F + LIGHT

where the asterisks represent an excited statě and F is thefluorescent acceptor (enhancer). The Cl- and Br-substituteddioxetanes give lower background and lower detection

1 'í

limits when compared with non derivatized ones '. Thenew dioxetanes contaín a modified hydrophobic adamantylgroup to prevent aggregation, and the background chemi-luminescence observed was 50 % lower than that of PPD.

2 . 5 . A c r i d a n s C h e m i l u m i n e s c e n c e

Recently another new substráte for horseradish peroxi-dase (HRP) has been produced: a stable trifluorophenyl-substituted acridan (Lumigen PS-3), which emits fotons at430 nm. The detection of HRP activity by PS-3 is based ona chemiluminescent process involving the enzymatic gene-ration of an intermediate acridinium ester. In the presenceof hydrogen peroxide and jo-iodophenol as enhancer, the

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reaction of the acridan substráte with the HRP label gene-rates thousands of acridinium ester intermediates ([AE]+)per minuté, which react with hydrogen peroxide, at alkalinepH, to produce the N-methylacridone in the singlet excitedstatě. This compound comes back to ground statě producinga sustained high-intensity chemiluminescence, accordingto the scheme:

3. Immobilized Flow Systems

The continuous flow formát of the analytical methodoffers greater possibilities than a single batch systém be-cause it leads to rapid and sensitive assays. The immobi-lized enzymes, on nylon coils or methacrylate beads, canbe ušed for many analyses and multichannel systems canbe developed. The analytical performances in terms ofdetection limit, linearity and reproducibility for the diffe-rent methods developed are reported in Table I. As shown,many analytes can be detected at pmol levels, with goodprecision and a wide range of linearity. Moreover, unlesshigh sensitivity is required, they reduce the analysis time toa few seconds and need smaller sérum specimens.

Fig. 1. Example of typical luminescent flow sensing device(manifold for pesticide chemiluminescent flow assay with one(A)or two (B) columns using immobilized enzymes)

Table IAnalytical performance of the immobilized luminescentenzymes

aAbbreviations: (B) bioluminescent bacterial systém on ny-lon, (L) bioluminescent firefly systém on nylon, (C) che-miluminescent luminol systém on nylon, (M) biolumines-cent firefly systém on methacrylate beads, (r-LM) recom-binant firefly luciferase on methacrylate beads, (r-LN)recombinant firefly luciferase on nylon, (imol.min" .1"

3 . 1 . Apparatus

The generál scheme of the systém is designed to be assimple and versatile as possible (Fig. 1). Using enzyme

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immobilized on nylon coils, the systém is schematicallycomposed of a sampler which permits insertion of thesample within two bubbles of air or directly after a suitabletime interval by a three-way valve, another two or threestreams for buffer or substrates supply, specific immobi-lized enzyme, and finally the luminescent enzyme(s) reac-tor inserted directly into the luminometer. In order toachieve reproducible and accurate measurements, the sam-ple must be separated from the working solution stream bytwo air bubbles to prevent axial diffusion caused by theconcentration gradient which can reduce reproducibility.

The beads (usually Eupergit) with immobilized en-zymes are generally packed into a small Plexiglas column(3 cm x 2 mm i.d.) and the column is located inside theluminometer in front of the photomultiplier window. Thesamé systém as ušed for coils can be employed for theEupergit column, except that the air is replaced with bufferin the second stream and the presence of air bubbles insidethe column is carefully avoided. The Eupergit column canbe also coupled with a nylon coil containing immobilizedenzymes (not luminescent), that can be placed before thecolumn, outside the luminometer.

3 . 2 . A p p l i c a t i o n s

Table I shows a list of analytes, which háve beendetermined using immobilized luminescent enzymes.

A typical example of these analytical systems is a mani-fold using bacterial luciferase for L-phenylalanine assay14

developed with two separate nylon coils: the first onecontains the specific L-phenylalanine dehydrogenase (L-PheDH) enzyme:

L-phenylpyruvate + NADH + H+ + NH4

and the second the bacterial bioluminescent enzymes. Thissystém allows reaching a detection limit of 0.5 (imol.F .

A continuous-flow systém was developed for the assayof magnesium (II) in sérum, drugs and beverages, makinguse of firefly luciferase (LUC) or recombinant luciferase(r-LUC) from Escherichia coli immobilized both on nyloncoil and on epoxy methacrylate15. The sample assay ratewas 20 samples.lr1 with no carry-over; 10 u.1 samples wereanalysed. The detection limit of the assay was 0.05 mmol.l"1

for LUC-nylon coil and 0.01 mmol.l"1 for LUC-Eupergitcolumn and r-LUC-nylon coil. No interference from ionspresent in the samples (Ca2+, Mn2 +, Fe2 +, Cu2+, Zn2 +,

Co2+) was found. The nylon-immobilized enzymes give t

a relatively high stablility (1-4 months), despite of a lowrecovery in terms of activity with respect to the solubleforms; Eupergit C gives better sensitivity and activity re-covery but lower enzyme stability (3 days-1 month).

The manifold showed on Fig. 1A has been ušed forcholine and/or phospholipase D assay16. Two columnsmanifold (Fig. 1B) successfully allowed determination of pes-ticides, like paraoxon and aldicarb17. These flow systems donot require thermostating, light emission being constant intherange 18-28 °C;however, this can be doně ifan accuratecontrol of temperature is needed, particularly when enzy-matic activity is measured16, e.g. phospholipase D. On theother hand, flow-rate variations do not influence light emis-sion over a sufficiently wide range. Variations in samplevolume obviously háve greater effects when a short coil isušed (50 cm) since saturation values are soon reached.Using a longer coil (e.g. 1 m or more), the light signálremains constant over a larger range of sample volumes. Ifhighly sensitive analytical performance is needed, the low-light imaging luminograph can be ušed18. This instrumentcan be also applied for batch enzyme activity assay19.

3 . 3 . M i c r o d i a 1 y s i s T e c h n i q u e

In vivo metabolite determinations can be performed byinserting „an artificial capillary" into a target area (brainarea, muscle, adipose tissue, blood, etc). The probe consistsof two concentric steel cannulas covered at the tip bya dialysing membráně (molecular mass cut-off around20,000); a physiological fluid is introduced through theinner cannula, flushes the inside of the membráně, andleaves via the outer cannula20. This systém allows samplingof endogenous substances from the extracellular space.

We applied the immobilized enzyme luminescent assayof lactate to the direct determination of this metabolite inischemic rat brain21. The microdialysis probe, with a 2 x0.5 mm dialysing membráně at the tip, was inserted into thenucleus caudatus-putamen of both normál and ischemicanaesthetized rats, connected to a microinfusion pump andcontinuously perfused at 2 (il.min"1 with Ringer solution.The recovery of lactate through the dialysis membráně,tested by in vitro experiments with standard solutions, wasaround 4-10 %. The perfused Ringer solution was collectedevery 5 minutes (10 p.1 total volume) and immediatelyanalyzed for lactate content. Results were in agreementwith the values obtained at the samé conditions by othermeasurement techniques, such as HPLC, which, however,require larger injection volumes.

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4. DNA and RNA Probes

We háve developed dot blot hybridization immuno-enzymatic assays with chemiluminescent substrates for thesensitive and quantitative detection of cytomegalovirus,B19 Parvovirus and plasmid pBR328 DNAs7-22. Digoxige-nin-labelled probfes were ušed and then visualized by anti-digoxigenin Fab fragments labelled with alkaline pho-sphatase or horseradish peroxidase8-9. Digoxigenin waspreferred as a nucleotide marker in order to avoid problemsthat may arise from the use of biotin-labelled probes de-tected with avidin systems in sera and because of its highspecificity and sensitivity similar to that of radiolabelledprobes, but without the potential health hazards, disposalproblems and instability associated with radioisotopes.

Light emission can be revealed both by instant-develop-ment photographic film as a qualitative test and by instru-ments like luminometer or luminograph as a reliable quan-titative assay in clinical samples. In recent years, the re-search and synthesis of improved chemiluminescentsubstrates has been matched by new developments in pho-ton imaging instrumentation such as ultrasensitive lumi-nographs based on an intensified charge-coupled device(CCD) video camera. This instrument allows the quantifi-cation of the light signál at the level of a single photon andlocalization of the chemiluminescent emissions on a targetsurface, such as tissue or membranes22. Spots of hybridizedDNA, cut out from nylon membráně, were placed intocuvettes containing the chemiluminescent mixture anddirectly analysed in a luminometer, and were revealed byALP-conjugated and by HRP-conjugated reagents.

Of the two labelling systems, the ALP systém, usingPPD as the chemiluminescent substráte, was more reliableand appliable. In fact, the samples can be stored dry at 4 °Cfor at least 1 week and it is not necessary to measure themimmediately like for HRP conjugates; the measurementscan be performed all together, not one by one because thelight emission is not an unstable flash but glows for severaldays. The results obtained with ALP detection were morereproducible: the relative standard deviation was about6 %, whereas that obtained with the HRP detection systémwas13 %.

The sensitivity of the chemiluminescent assay is com-pared with that of the colorimetric one in Table II. Moreo-ver, for membrane-based applications (Southern blottingand DNA sequencing) the Cl-derivatives give, in comparisonwith PPD, faster kinetics, permitting faster exposures, andsharper images of the bands obtained for DNA sequencing

blots. For both systems, a key step was the use of the samékind and batch of nylon membráně, because some types arevery highly charged and can cause a high background; toavoid this, each batch must be pre-tested.

Table IISensitivity of colorimetric and dioxetane ALP chemilumi-nescent photographic test detection of viral and plasmidDNAs using different concentrations of digoxigenin la-belled probes

5. Conclusion

The variety of the assays reported here displays the greatversatility of the luminescent detection systems. The mainadvantages are the high sensitivity and specificity, thatreduce to the minimum the sample treatment. Immobilizedsystems allow to reduce greatly the cost per assay, on theother hand their preparation requires a good expertise,especially in the surface activation step on nylon tubes.

The bioluminescent flow sensors based on enzymesimmobilized on nylon tubes and Eupergit columns specifi-cally assay several analytes and enzymes activities (Tab-le I) and are simple, require only minor modifications ofa commercial detector and allow the analysis of about20-30 samples per hour. Therefore the flow bioluminescentmethods appear highly competitive with other meťhodssuch as radioimmunoassay, enzyme immunoassay, highperformance liquid chromatography, and fluorimetry whichrequire radioactive materials, separation steps, sample ma-nipulation or expensive equipment.

When the flow sensors are ušed coupled with microdia-lysis probe there are several advantages:- the reaction takés pláce in few minutes allowing con-

tinuous analysis,- sensitivity is in the order of pmoles,- the microdialysis probe allows biological specimens to

be drawn without proteins or macromolecules.

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This approach can be extended to the analysis of otheranalytes which need to be detected continuously, such asduring therapy monitoring or in emergency care units.

The use of chemiluminescent dot blot DNA and RNAhybridization techniques suggest that chemiluminescentsubstrates can be considered useful tools for the detectionof nucleic acid hybridization reactions because of their highsensitivity and specificity. In the čase of in šitu hybridiza-tion assays the best performance can be obtained using newinstruments, such as videocamera luminograph with com-puter collecting data19.

This work was supported by grantsfrom MURST (Min-istero della Universita e della Ricerca Scientifica e Tec-nologica), CIRB (Centro Interdipartimentale di RicercheBiotecnologiche) and the European Community ResearchProjed No. CÍP A CT 94-0147.

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S. Girotti3, E. N. Ferri3, S. Ghinia, F. Fini3, M.Musianib, G. Carreac, A. Rodad, P. Rauche ("Institute ofChemical Sciences, University of Bologna, Institute ofMicrobiology, University of Bologna, cInstitute of Hor-mone Chemistry, CNR, Milano, ^Department of Pharma-ceutical Sciences, University of Bologna, Italy, e Institute ofChemical Technology, Prague): Luminescent Techni-ques Applied to Bioanalysis

The principle of luminescent systems (firefly and bac-terial bioluminescence, luminol, dioxetane and aeridanschemiluminescence) is deseribed. The bio- and chemilumi-nescent reagents can be employed as highly sensitive and,together with immobilized enzymes, also specific detectionsystems in bioanalytical assays both by using batch deter-mination and continuous flow systems. An example ofversatile manifold (flow sensing device) is shown. A list ofpossible bioanalytical applications is given.

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