CN102121056B - Microbial detection and quantification - Google Patents

Abstract

A method for semi-quantitatively or quantitatively detecting the presence of a microbe in a sample is provided. The method utilizes a test dye that undergoes a detectable color change in the presence of one or more microbes. For example, in one embodiment, the test dye is a solvatochromic dye (e.g., Reichardt's dye) that responds to differences in polarity between microbe components (e.g., cell membrane, cytoplasm, etc.) and the environment outside the cell. Alternatively, other mechanisms may be wholly or partially responsible for the interaction between the dye and the microbe, such as acid-base reactions, redox reactions, and so forth. Regardless, the color of the test dye may be compared to the color of a control dye, wherein the color of the control dye corresponds to a known microbe concentration.

Description

Microorganism detection is with quantitative

Related application

The application is the part continuation application of the PCT/US2004/042461 international application of submission on December 16th, 2004, the right of priority that No. the 10/737th, 574, the U.S. Patent application of this international application requirement submission on December 16th, 2003.

Background of invention

The present invention relates to detect method and the product of microorganism such as bacterium, yeast, mould and virus.

In our daily life, we are exposed to the body surface that is subjected to microbial contamination unawares, and this can cause diseases.There are some researches show, concrete bacterial contamination " focus " comprise Public telephone, door handle, doctor's waiting room and children nurse the toy in the place, dried hand with the towel that uses in hot air dryer, the kitchen and sponge, carry out daily patient care medical personnel hand and mix raw meat and the making food surface of vegetables and the crossed contamination of cutter.

Singly in the several local bacterial contamination outburst events that occur of the U.S., just caused some children and the elderly death and other people to fall ill recently.The microbial contamination of food also is global subject matter.Salmonellas, intestinal bacteria and other food infect bacterium and cause unnumbered case every year.That acute symptom comprises is nauseating, vomiting, abdominal colic, diarrhoea, fever and headache.Then may be exactly chronic consequence after the acute symptom outbreak.Because the crossed contamination meeting of body surface causes that bacterium from interior, fish and poultry to the transfer of not boiling food such as vegetables, easily detects bacterium and will have very big benefit in the ability of the lip-deep existence of making food.

Equally, to the detection of the microorganism of harmful level, be very important for the health that keeps each family and human consumer in the food processing enterprises.In food-processing industry, bacterial monitoring is vital.No matter the processing of nearly all food from the meat Package to the cheesemaking, all relate to monitoring microorganism level, to guarantee the security of food supply.

The disaster that microbial contamination brings is not limited only to foodstuffs industry.Recently decades witness sharply increasing of " superbacteria (superbug) ", this problem is just concentrated and is appeared in hospital and the health institution.The deficiency of antibiotic excessive use and hospital's cleaning has expedited the emergence of methicillin-resistant staphylococcus aureus (S.aureus) (MRSA) and clostridium difficile (Clostridiumdifficile) and Vancomycin-resistant Enterococcus and other gram negative bacillus (Dancer, 2004).The recently report of British Broadcasting Corporation (BBC) is mentioned, and the annual estimation of MRSA seizes 5000 life.The report article asserts that then " cleaning remains main patient's concerned issue, and MRSA is a more and more serious problem." non-responsiveness effect of many patients in the hospital of justing think, it is higher that therefore the risk that infects occurs, the threat that the vicious bacterium in the hospital environment is caused can become all the more dangerous.

Many reports and research are arranged in the problem of inquiring into hospital's cleaning and hospital's infection control.

Equally, known mould such as ergot can grow in some cereal such as rye, and they have potential hazardness owing to can producing the toxic alkaloid similar to sphacelic acid.The spore that known aspergillus niger (Aspergillus niger) and other mould produce can cause anaphylaxis and increase the weight of respiratory passage diseases such as asthma.If aspergillus niger begins growth on moist wall or in the conditioning unit in family or commercial building, will be a problem especially.

Some yeast such as Candida albicans (Candida albicans) can represent another kind of disagreeable microorganism.White mouth and vagina yeast infection with Candida albicans and infant diaper rash, children and non-responsiveness adult connects.Yeast also can infect buccopharyngeal area and the gi tract of health.

Present method of detecting bacterium relates to slave unit acquisition surface sample.In the food-processing environment, equipment may be meat cutting machinery, and in making food environment such as restaurant or in family, the surface may be desk, chopping block, refrigerator inside or working-surface.Then sample is incubated overnight to grow culture.It is to allow sample grow under suitable temperature and humidity on the agar plate that overnight growth is cultivated, and makes bacterial growth and breeding must be enough to macroscopic bacterium colony greatly to forming.At incubation specific time and allow bacterial colony grow out after, by trained technician's hand inspection agar plate sample and estimate colony-forming unit (CFU).This method spends some height, and upper greatly hysteresis of time; Polluted product may betransported away or people have been exposed to existing microorganism in the middle of retardation time.

As from the foregoing, the rapid detection of being convenient to harmful microbe method and product need to be arranged.

Summary of the invention

Be the aforementioned difficulties that reply those skilled in the art run into, we have developed a kind of indication composition, but it comprises moving phase and the obviously sensitive colorant (colorant) of microorganism of change detected can occur in the presence of microorganism.Described composition can be applied to the surface to show existing of microorganism.Moving phase can be sterilizing agent.Colorant provides macroscopic colour-change in the presence of microorganism.Flowability can be liquid or gel, and colorant can be dyestuff (dye).In some embodiments, the concentration of the degree of colorant change color and microorganism is proportional.In other embodiments, the quantity of existing microorganism is proportional with the amount of the colorant that colour-change occurs.

The example of suitable dyestuff comprises that merocyanine dye, 4-[2-N-replace-1,4-hydrogenated pyridine-4-subunit) ethylidene] hexamethylene-2,5-diene-1-ketone, red pyrazolone dye, azomethine dyes, indoaniline dyes, diaza merocyanine dye, the zwitter-ion dyestuff as an example of the Reichardt dyestuff example and other dyestuff and their mixture.Particularly suitable dyestuff is the zwitter-ion dyestuff, and wherein zwitter-ion is included in adjoining in the middle of the π-electron system of formation dyestuff chromogen.As if the another kind of dyestuff that is particularly useful as microorganism indicator be merocyanine dye.

Also dyestuff can be applied to the surface with the form of solvent-based or group water solution and allow its drying, stay the dried residue of the dye solution that applies.Dried residue is the meeting variable color when touching microorganism, therefore can be used on packing as on the facial tissue box, on carry-on medical instruments such as gloves and on other surface, these surfaces make with dyestuff first before use, but so that later indicator microoraganism pollution condition.Surprisingly, the inventor finds when these dyestuffs being applied to the surface upper and when allowing its drying, all the microorganism detection ability of coating is had significant impact in order to the solvent of making coating (coating) and used additive such as hydroxypropyl-beta-cyclodextrin and tensio-active agent.

Found that hydroxypropyl-beta-cyclodextrin can the luminance brightness of Effective Raise colorant after it is coated on paper handkerchief or the similar Wiping material.Although do not want to be bound by theory, we think that still the color of dyestuff is improved because of the crystallization that the cyclodextrin derivative that adds suppresses dyestuff.Other pharmaceutical chemicals can be joined in the cleaning piece (wipe), read the result to help prevent because of the false positive that existence was caused of SYNTHETIC OPTICAL WHITNER (found that it can disturb dyestuff).

The lateral flow device that combines microorganism indication colorant is also included within the instruction content of the present invention.These devices have the film with detection zone and check plot, and wherein detection zone responds and variable color to the existence of bacterium, and the check plot still keeps original dye colour to show that test is true(-)running.

This paper also describes the lip-deep method of microorganism of inspected object, and the solution that this method will contain the sensitive colorant of microorganism is applied to the surface, but and observes the obvious change detected that shows that microorganism exists whether occurs.

Further feature of the present invention and aspect have more detailed discussion hereinafter.

The accompanying drawing summary

At the remainder of this specification sheets, more specifically set forth detailed and enforceable disclosure of the present invention towards those of ordinary skills, comprise preferred forms, relate to the following drawings in the elaboration:

Fig. 1 is the schematic diagram of five kinds of basic bacterial cell shapes.

Fig. 2 is the schematic diagram that bacterial cell is arranged.

Fig. 3 is a kind of structure of merocyanine dye.

The method of a kind of synthetic merocyanine dye of Fig. 4-5 explanation.

Fig. 6 A-D is with displaying the schematic diagram that shows microbial contamination that chicken is done.

Fig. 7 A-G is the schematic diagram that side by side contrast shows microbial contamination and cleaning situation.

Fig. 8 A-D is the schematic diagram that shows microbial contamination of doing with the different concns bacterium.

Fig. 9 A-E is with showing of doing of bacterium and indicating dye titration and the quantitative schematic diagram of microbial contamination.

Figure 10 A-C is the schematic diagram that shows the microbial contamination on the computer keyboard.

Figure 11 A-D is with having and not having the solution of tensio-active agent to show the schematic diagram of microbial contamination.

Figure 12 A-F is the schematic diagram that shows the microbial contamination indication speed that depends on solvent.

Figure 13 A-C is the schematic diagram that shows microbial contamination in colorant is dried to situation on the base material (substrate).

Figure 14 is illustrating of embodiment 30 acquired results, and wherein Δ E is to the staphylococcus glucose coccus mapping of concentration known.

Figure 15 is illustrating of embodiment 30 acquired results, and wherein Δ E is to Pseudomonas aeruginosa (P.aeuruginosa) mapping of concentration known.

Figure 16 is illustrating of embodiment 30 acquired results, and wherein Δ E is to the intestinal bacteria mapping of concentration known.

Figure 17 is the top view that can be used for an embodiment of lateral flow determinator of the present invention.

Figure 18 is the reflection molecule relates to transition of electron in the middle of the molecule to visible Optical Absorption schematic diagram.

Nonexpondable reference character means to represent identical or similar characteristics or key element of the present invention in this specification sheets and accompanying drawing.

Detailed Description Of The Invention

The present invention relates to the detection of bacterium and other microorganism, term used herein " microorganism " is understood to include bacterium, fungi (such as yeast and mould) and virus.

There are thousands upon thousands kinds of different types of bacteriums to exist.Some kind difference is very little, needs well-trained personnel could identify them.Also some kind is easy to just identify in habit with difference is very large in appearance.As not considering tiny difference, then most of bacteriums can be classified by five kinds of elementary cell shapes shown in Figure 1.Fig. 1 from left to right, shape is respectively circle or coccus, shaft-like or bacillus, spirrillum or spirilla, comma shape or vibrios and thread.

Except the shape difference, their cell arrangement is also different, and Fig. 2 from left to right is respectively diplococcus, suis and staphylococcus.For example, some coccuses often become double focusing collection (diplococcus).Other then arranges chaining (suis).Also have other to arrange bunchiness (staphylococcus).Diplococcus has been notified and has caused pneumonia.Suis is then often relevant with " septic pharyngitis (strep throat) ".Staphylococcus is familiar with by everybody because of its effect in the foodborne illness of " staphylococcal infections " and some type.

Bacterium also has certain difference aspect big or small, but single bacterium mean size be about 1/25,000 inch (1 inch=2.54cm).In other words, 25,000 side-by-side 1 inch straight lines that also just only account for of bacterium.One cubic inch of bacterium that is enough to hold 9,000,000,000,000 mean sizes, namely everyone can share about 3,000 bacteriums on the earth.

Although based on the modern molecular biology concept theoretical basis of the careful classification of bacterium is had a lot of discussions, but for microbial project teacher, Fast Subdivision is take Gram-reaction (dyeing process that bacterium is classified) and morphology as the basis.

Gram positive bacterium can keep violet staining in the presence of alcohol or acetone.They comprise following important genus: actinomyces (Actinomyces), bacillus (Bacillus), genus bifidobacterium (Bifidobacterium), Cellulomonas (Cellulomonas), fusobacterium (Clostridium), corynebacterium (Corynebacterium), micrococcus sp (Micrococcus), Mycobacterium (Mycobacterium), Nocardia (Nocardia), Staphylococcus (Staphylococcus), streptococcus (Streptococcus) and streptomyces (Streptomyces).The bacterium of some gram positive bacteriums, particularly corynebacterium, Mycobacterium and Nocardia, even in the presence of acid, also dyestuff can be kept.They are called as acid-fast bacterium.

Gram negative bacterium can not keep violet staining in the presence of alcohol or acetone.They comprise following important genus: genus acetobacter (Acetobacter), Agrobacterium (Agrobacterium), Alcaligenes (Alcaligenes), wrap special Bordetella (Bordetella), Brucella (Brucella), campylobacter (Campylobacter), Caulobacter (Caulobacter), enterobacter (Enterobacter), erwinia (Erwinia), Escherichia (Escherichia), Helicobacterium (Helicobacterium), legionella (Legionella), eisseria (Nesseria), Nitrobacter (Nitrobact), pasteurella (Pasteurelia), pseudomonas (Pseudomonas), rhizobium (Rhizobium), rickettsiae (Rickettsia), salmonella (Salmonella), Shigella (Shigella), Thiobacillus (Thiobacilus), Wei Rong Shi Coccus (Veiellonealla), Vibrio (Vibrio), xanthomonas (Xanthomonas) and Ye Erxin Bordetella (Yersinia).

Bacterial cell membrane is made of the double-layer of lipoid of lipopolysaccharides usually.Difference (being cell walls) is arranged between the cytolemma of gram negative bacterium and gram positive bacterium.The cell wall structure of gram negative bacterium is thinner, have obvious which floor.Outer and typical trilaminar structure combination is more as cytoplasmic membrane.

The main component of gram-negative bacterial cell wall is lipopolysaccharides.Also there are in addition phosphatide, protein, lipoprotein and a small amount of peptidoglycan.Lipopolysaccharides is comprised of with polysaccharide part (moiety) repeating unit that is connected thereon core area.Have certain composition relevant with interior toxicity activity in the cell walls of most of gram negative bacteriums, this interior toxicity activity then is related to the pyrogen reaction of gram-negative bacterial infections.Carrying the specific basic substance of O antigen of these bacteriums on the side chain.The chemical constitution of these side chains (being related to each composition and different carbohydrate arranges) has determined the in other words character of O antigenic determinant of O antigen, and this is the very important means of on serology many gram negative bacterium kinds being classified.In many cases, confirmed that the very different kind of some bacterium ownership but can produce the reason of strong serological cross reaction, just be that they with the part of chemically similar carbohydrate part as their lipopolysaccharides side chain, have approximately 30 repeating units usually.

The feature of gram positive bacterium is them with peptidoglycan and polysaccharide and/or the teichoic acid part as their cell wall structure.Peptidoglycan also claims murein sometimes, is that many polysaccharide chains are by the crosslinked heteropolymer that forms of small peptide.

The basis of murein is many chains that N-acetyl-glucosamine residue alternately and-acetylmuramic acid residue are formed by connecting by β-Isosorbide-5-Nitrae key.Teichoic acid is unique material of bacteria cell wall.These chains are crosslinked together by the short polypeptide chain that L-and D-amino acid form.In gram negative bacterium, peptidoglycan is simple in structure, and is all more consistent in great majority belong to, and in gram positive bacterium, its structure and composition difference is very large.In general, peptidoglycan is multilayer.Also theory on the books, some microbe groups are some little difference on forming.Therefore, in mycobacterium and Nocardia, the oxidized form N-glycolyl of the N-acetyl moiety of teichoic acid partly replaces.All the possibility difference is very large on the amino acid composition of crosslinked polypeptide and trunk polypeptide for Different groups.These difference have consisted of the basis of the classification of these bacteriums.

Molds and yeasts belongs to fungi.Although a lot of moulds and fungi are useful to the mankind, some has pathogenicity bo, can discharge the harmful fungoid toxin and causes poisoning or dead.Yeast also can cause infecting, the yeast vaginitis that is widely known by the people that the chances are most.

Zygomycota (Zygomycota) is a class fungi, comprises mould and other mould of ankerstoke, exists symbiotic relationship with plant and animal.These moulds can be merged and be formed solid " zygospore ".Ascomycota (Ascomycota) is another kind of fungi, comprise yeast, powdery mildew, black mould and bluish-green mould and some can cause for example kind of Dutch elm disease, scab of apple and ergot of disease.Made up sexual propagation and vegetative propagation the life history of these fungies, mycelia is subdivided into the porous wall that allows nucleus and tenuigenin to pass through.Imperfect fungi door (Deuteromycota) is another class fungi, and various fungies or the Basidiomycetes (Basidiomycota comprises most of mushrooms, pore fungus and Lasiosphaera nipponica(Kawam.)Y.Kobayasi) that can not certainly be fit to above-mentioned classification all are included in the middle of this type of.These deuteromycetes (deuteromycetes) had both comprised the kind of making cheese and penicillin, also comprised pathogenic kind, as caused the kind of tinea pedis and ringworm.

In recent years, dyestuff is having outstanding growth in the use of biomedical sector aspect research interest and the technical significance.Dyestuff is in many fields of for example analytical biochemistry, medical diagnosis, even in the treatment and prevention of disease, all used.It is essential that the color of dyestuff is used for some, described application detects (United States Patent (USP) the 5th from being used for light splitting, 036, No. 000) and the body fluid analysis thing measure (No. the 0 250 700, European patent) simple organic reaction to the high resolution imaging technology (Motohashi that is used for lesion detection, Med.Res.Rev., 11,239,1991).Dyestuff also can be used for the treatment of disease (United States Patent (USP) the 5th, 468, No. 469) clinically.Photodynamic therapy (Sedlacek, " The change in research for the therapy oftumors ", Chimia, 45,52,1991) successfully be used for the treatment of the cancer of some kind, such as the malignant tumour of skin, head, neck, lung and esophagus.Other therapeutic is used and is related to the antiviral of dyestuff and kill the bacterium performance.Dyestuff also is crucial material in histology, biological mark and these key areas of biological probe.Relevant technology is very complicated, need to dye, washing and Cross reaction (Blum, Photodynamic action and disease caused by light " Reinhold, New York, 3,1941).

The inventor finds, can prepare microorganism indication spray and develop the fast microbiological quantivative approach with specific colorant.The potential application of this technology includes but not limited to detect the microorganism on the following solid surface: sales counter end face, hand, medical area, bathroom, bedrail, medical facilities, operating table, vessel, kitchen, food, making food surface, food processing plant, door handle, phone and computer keyboard.Whether this coloured dye coatings, spray or solution are sensitive to bacterium and other microorganism of harmful level, and its colour-change is being served as visual indicator means, effective in order to the cleaning and/or the purification that confirm the surface.

Requirement to pointing technology is quite strict, because used dyestuff must be quick to gram positive bacterium strain and gram negative bacterium strain Turin.Dyestuff should be able to interact with microorganism or microbe metabolite rapidly.For the multifunctionality of maximum, it is sensitive that dyestuff is also tackled other microorganism such as yeast and mould.

As previously mentioned, dyestuff has been used as the staining agent of cell and Bacteria Identification for a long time.Dyeing solution and cell or bacterium react or are preferably kept by cell or bacterium, thereby help identify (Johnson, 1995) by the contrast that improves between it and existing background or other composition.Usually, staining agent must be applied to the surface, then remove excessive staining agent by vibration or rinsing, to highlight existing of microorganism.The inventor has any report about colorant that can variable color when being exposed to microorganism or occuring to interact with microorganism before not hearing.

Solvation variable color (solvatochromism) phenomenon may be to cause the reason of the colour-change of seeing, but the inventor does not want to be subject to a concrete theoretical restriction.Colour-change will occur in the solvation chromotropic dye when molecule environment such as solvent polarity and/or hydrogen bonding tendency appearance variation.For example, dyestuff color in polarity environment such as water may be blueness, but may be yellow or red in such as the solution that is rich in lipid in nonpolar environment.The color that this " suitable dye " produces depends on the ground state of dyestuff and the molecular polarity difference between the excited state, and this has more comprehensively hereinafter discusses.The Reichardt dyestuff is chosen as research model dyestuff.

The inventor wonders, by the polarity difference between some cellular constituent (such as cytolemma, tenuigenin etc.) and the outside is responded, whether some solvation chromotropic dyes can be used for detecting microorganism.The inventor finds, when microorganism and some are coated in these dyestuffs on the base material (such as paper handkerchief) and contact, really observe colour-change---not merely be colour-change, and in most of the cases dyestuff is to decolour in the zone that is touched by bacterium.Make the inventor surprised be, further also prompting of research, the mechanism in the middle of this also not exclusively is attributed to the solvation metachromatism.In fact, the present inventor reports at this, and what exceed that they expect is that they find the following fact:

I) can be related with being undertaken by the amount of the dyestuff of bacterium or the decolouring of other microorganism and the concentration of the microorganism that is exposed to dyestuff, prompting the method is quantivative approach and qualitative method,

The scope of the microorganism that ii) can be detected comprises Gram-positive and gram negative bacterium, yeast and mould,

Iii) tested dyestuff can be used as the form of dry film coating or as adding the form that contains the solution in the bacterial liquid to or use as the form of spray-type detection system,

Iv) when using as the form of the dry coating on for example paper handkerchief or the enamel surfaces, the character that applies according to this solvent of this dyestuff makes a significant impact the performance (contrast and sensitivity between bleaching time, decolouring zone and the non-decolouring zone) that detects with dyestuff

V) when using with the form of the dry coating on the paper handkerchief for example, being included in additive in the coating with dyestuff can affect the performance that detects with dyestuff (decolouring apply, decolour contrast and sensitivity between regional and the non-decolouring zone).For example, hydroxypropyl-beta-cyclodextrin can strengthen the performance that detects with dyestuff,

Vi) decolouring of bacterial these dyestuffs can reverse with highly basic.

Although the solvation metachromatism can be facilitated viewed colour-change, these observationss may also accord with other and seem reasonable mechanism.For example, these observationss may meet also that certain type of acid-alkali interacts or certain type to alpha proton reaction, the dye colour that this reaction can be facilitated bacterium to exist and cause changes.The inventor does not yet have complete ruled it out, and namely when some dyestuff was exposed to multiple-microorganism, the reaction of redox type also may be facilitated the colour-change of perceiving.Other factors may also can be facilitated the colour-change of viewed some dyestuff in the presence of microorganism, and for example, there are interaction in the part of possible cytolemma and some dyestuff and cause colour-change.Also having a kind of possibility is that height systematism acid moieties (moiety) on the bacteria cell wall may can make some indicating dye protonated, causes losing color.

The inventor has found a kind of unusual and present not construable phenomenon also, and utilizes this phenomenon to develop to can be used for detecting and the quantitative method of multiple-microorganism.

In general, with regard to the visual detection of colour-change, " color " is a kind of modality of sensation, produces when human eye mechanism perceives the existence of the various wavelength light that object reflected or launched in the visual field or do not exist.The cone cell to visible spectrum specific region sensitivity by three types carries out spectroscopic analysis to the light that enters eyes.Then again by retinal neurons, optic nerve neurone with look cortex and process, the result experiences the sensation of color from the stimulation of these cells.Give mechanism (such as absorption, emission, fluorescence, phosphorescence, refraction, diffraction etc.) although exist several colors, suitable research focus is limited to absorptivity color (absorptive color).In other words, the present invention relates to because of the colorific dyestuff of the light that absorbs some wavelength.

Because the human eye function mode, the color that perceives normally object the complementary color of color corresponding to light-absorbing wavelength.Look like red object when for example under white light, watching, in fact absorb the light blue light of 490-500nm wavelength region in selectivity.Similarly, seem that under white light the object of yellow is in fact at the blue light that absorbs the 435-480nm scope.

Molecule relates to transition of electron in the middle of the molecule to visible Optical Absorption, and causes the generation of excited state.According to following Planck relation, capacity volume variance is determining light-absorbing wavelength between the ground state of molecule and the corresponding excited state:

E＝hv

E=energy in the formula, h=quantum of action, v be the frequency of light-absorbing photon, as follows with the relation of wavelength X and light velocity c:

v＝c/λ

Figure 18 has illustrated transition of electron.

The energy of the photon that obviously, absorbs and the wavelength of photon are inversely proportional to.Therefore, the photon of blue light (435-480nm) has than the higher energy of gold-tinted (580-595nm).So when watching under white light, the color of the dyestuff in the solution or on the object allows that by the ground state and first of dye molecule the transition the subject of knowledge and the object of knowledge between excited state determines.

The extinction part of dyestuff usually is called the chromogen of dyestuff.Chromogen comprises chromophoric group and it the conjugated system that connects.Chromophoric group is the main group that produces the color of dyestuff, for example azo group in the azoic dyestuff, the polyenoid group in the carotene, the carbonyl group in the anthraquinone dye.Also have many other chromophoric grouies.Auxochromous group affects color and the intensity of dyestuff by acting on the conjugation chromogen.Auxochromous group can with or can be not and the chromogen conjugation.For example, the amino by for example phenyl ring and azo group (chromophoric group) conjugation can form amino azo chromogen.The amino auxochromous group of conjugation can be offset the absorption band of azo group to longer wavelength, increases the intensity of absorption band.But, have a mind to sulfonic acid group added in the amino azo chromogen conjugation does not occur, but sucting electronic effect makes and absorbs skew to longer wavelength.

The example of the dyestuff that ground state polarity is larger than excited state polarity is merocyanine dye 1 as follows.The canonical structure 1 of the charge separation of left-hand side be ground state mainly facilitate structure, and dexter canonical structure 1 ' be first excited state mainly facilitate structure.

As follows indigo 2 is that ground state polarity is significantly less than the example of the dyestuff of excited state polarity.The canonical structure 2 of left-hand side be this dyestuff ground state mainly facilitate structure, and dexter canonical structure 2 ' be excited state mainly facilitate structure.

Can comprise that above-mentioned dyestuff and Reichardt dyestuff, merocyanine dye, zwitter-ion dyestuff (wherein form positive charge and negative charge are included in and adjoin in the middle of the π-electron system), 4-[2-N-replace-1 for implementing suitable dye of the present invention, 4-hydrogenated pyridine-4-subunit) ethylidene] hexamethylene-2,5-diene-1-ketone, red pyrazolone dye, azomethine dyes, indoaniline dyes, diaza merocyanine dye and their mixture.

Merocyanine dye belongs among " Colour and Constitution of Organic Molecules " Academic Press (London) 1976 donor discussed-simple acceptor chromogen Griffiths classification, and wherein carbonyl serves as electron acceptor moiety.This electron acceptor(EA) conjugation is to the electron-donating group that can supply with electronics such as hydroxyl or amino.Merocyanine dye is relatively wide dye, and this class dyestuff comprises structure 3, and the nitrogen-atoms that wherein comprises in the heterocyclic system serves as donor, and n can be and comprises any round values of 0.Merocyanine dye has charge separation (zwitter-ion) resonance form.

Acyclic merocyanine dye also is known, comprises vinylalogous amide.

Studied merocyanine dye and made silver halide have photosensitive ability to some wavelength of light, in photographic film.Many merocyanine dye structures are known.Structure 4-14 shows several limiting examples of merocyanine dye.Attention can draw the resonance structure of charge separation for every kind of these dyestuff.Document proposes, and (zwitter-ion) form of charge separation is the important structure of facilitating of dyestuff ground state.

Wherein R can be methyl, alkyl, aryl, phenyl etc.

The zwitter-ion chromogen

Can prepare and forever be some dyestuff of zwitterionic form.That is to say, these dyestuffs have the permanent charge that relates to the π-electron system, can not obtain the neutral resonance structure of chromogen.This dyestuff comprises Reichardt dyestuff 15, and it meets general structure 16.

Except the Reichardt dyestuff, with the solvation variable color pyridine of suitable negative charge

The other example of N-phenolic acid trimethyl-glycine (pyridinium N-phenolate) dyestuff is provided by following structure 16-21:

Wherein R be hydrogen ,-C (CH ₃) ₃,-CF ₃Or C ₆F ₁₃

Non-limiting structure 24-32 in addition can comprise following structure, and they meet general structure 23:

Wherein X can be hydrogen, carbon, nitrogen, sulphur.

The amount of dyestuff must be enough so that the variation of generation color when contact with microorganism, and this variation to be naked eyes perceive, this depends on the sensitivity of dyestuff.Discovery is in anhydrous, enough amount of dye are usually between 0.001 to 20 weight percent, in some embodiments between 0.01 to 10 weight percent, in some embodiments between 0.05 to 5 weight percent, in some embodiments between 0.1 to 3 weight percent.Colour-change occurs very soon, does not depend on concentration and the type of microorganism.

Composition comprises the sensitive colorant of aforesaid microorganism and moving phase.Belong to " moving phase " and comprise the liquids and gases that can be used as pigment carrier.Although can use any effective carrier, find that acetonitrile, tetrahydrofuran (THF), dimethylbenzene, formaldehyde (for example dimethyl formamide) and alcohol (for example methyl alcohol, ethanol, n-propyl alcohol and Virahol) are suitable carriers.Moving phase can also be sterilizing agent or kill bacteria composition.

The colorant dyestuff can be the form of liquid, and this liquid can spray or wipe on the surface, with existing of indicator microoraganism.The liquid that contains dyestuff can be applied on the surface, and allow the liquid dried that applies, form the dyestuff dried residue, in order to be exposed to later on microbial contamination.When being exposed to microorganism, dried residue meeting variable color, thereby the existence of indicator microoraganism.According to the present invention, colour-change may occur very soon.For example, chromogen can be less than approximately just beginning variable color in 30 minutes, in some embodiments less than approximately 5 minutes, in some embodiments less than approximately 1 minute, in some embodiments less than approximately 30 seconds, in some embodiments less than approximately 10 seconds.

The indicating means that this use solution applies the dried residue of dyestuff can be used on the solid surface, for example packing material such as facial tissue box, stickers (sticker), paper, toilet paper, carry-on medical instruments such as surgical glove, operating coat and drape (drape), face shield, head-shield is such as circle cap (bouffant cap), operating cap and scarf, latex examination gloves and surgical glove, footwear such as shoe cover, boots cover and slippers, wound dressings, bandage, sterilization scarf (sterilization wrap), handkerchief, clothes such as lab-gown, the working suit that connects trousers, apron and jacket, patient's bed clothes, stretcher and cradle coverlet, the making food scarf, wipe bowl dish sponge, cloth, door handle, phone, computer keyboard, computer mouse, pen, pencil, notepad, the washroom handle, wound dressings, bandage and toy are (for example at doctor's waiting room, the day care place).

Therefore, the solvation chromotropic dye base material that can apply thereon comprises cleaning piece and may be exposed to for example other article of above-mentioned bacterium.The solvation chromotropic dye also can be incorporated into to check in the washing lotion or white cream of microbial contamination of hand.Dyestuff also can be incorporated into sponge or dish towel, pollutes with caution.

Be suitable as cleaning piece and comprise any base material that is used as traditionally cleaning piece with the base material with the colorant coating, comprise film, woven cloths and non-woven, fibrous substrate such as toilet paper, paper handkerchief and common formation (coform) material, airlaid material, bonding carded web (web) etc.The nonexcludability example of relevant base material can be at United States Patent (USP) the 4th, 775, and No. 582, the 4th, 853, No. 281, the 4th, 833, No. 003 and the 4th, 511, find in No. 488, these patents have all transferred Kimberly-Clark Corporation.

Non-woven can be according to becoming the methods such as net method and carding method to make such as spunbond one-tenth net method, melt blown webbing method, airlay method, sticking solution.Non-woven can be by thermoplastic resin, includes but not limited to that polyester, nylon and polyolefine make.Alkene comprises ethene, propylene, butylene, isoprene etc. and their combination.

" spun-bonded fibre " is small diameter fibers, its formation method is as a plurality of small from spinning nozzle of filament with molten thermoplastic, usually circular kapillary is extruded, then by make the United States Patent (USP) the 4th of the diameter fast reducing of extruding filament: Appel etc. such as following patent, 340, No. 563, No. the 3rd, 692,618, the United States Patent (USP) of Dorschner etc., the United States Patent (USP) the 3rd of Matsuki etc., 802, No. 817, No. the 3rd, 338,992, the United States Patent (USP) of Kinney etc. and the 3rd, 341, No. 394, the United States Patent (USP) the 3rd of No. the 3rd, 502,763, the United States Patent (USP) of Hartman etc. and Dobo etc., 542, No. 615.Spun-bonded fibre is collected the surface and was not usually had tackiness when upper when being deposited on.Spun-bonded fibre is normally continuous, and mean diameter (mean values of at least 10 samples) is greater than 7 microns, and is preferably approximately between 10 to 20 microns.

" melt blown fiber " refers to following fiber: with molten thermoplastic as melting silk thread or filament by a plurality of small, during usually circular pattern kapillaries (die capillary) are expressed into convergences, the gas (for example air) of heat flows at a high speed, usually, this gas stream subtracts the filament of molten thermoplastic carefully, their diameter is reduced, can reach the primitive fiber diameter.Afterwards, melt blown fiber is carried by high velocity gas stream and is deposited on the collection surface, forms the melt blown fiber fibre web of random scatter.This method is such as open in No. the 3rd, 849,241, the United States Patent (USP) of Butin etc.Melt blown fiber is primitive fiber, can be continuous or discontinuous, and usually mean diameter is less than 10 microns, and usually has tackiness when go up on the surface when being deposited on to collect.

Term used herein " forms altogether " and refers to this method, and it is arranged at least one molten blowing mould head (diehead) on the next door of skewed slot, and fibre web is when can add other material by skewed slot when forming like this.These other materials can be paper pulp, high absorbing particles, natural polymer (for example artificial silk or cotton fibre or other fibrous material) and/or synthetic polymer (for example polypropylene or polyester) fiber, and wherein fiber can have staple length.Form altogether method at No. the 4th, 818,464, commonly assigned United States Patent (USP) (Lau) and the 4th, 100, in No. 324 (Anderson etc.) explanation is arranged.The fibre web of producing by common formation method is commonly called common formation material.

The bonding carded web is made from staple fibre, and staple fibre is carried by carding apparatus, and this device is broken up staple fibre and arranged along machine direction, forms usually take the nonwoven web of machine direction as orientation.Fibre web one forms, and just with in several methods such as powder bonded method, pattern mull technique (pattern bonding), the mull technique of drying and the ultrasonic bonding method one or more it is bondd.

In the airlay method, have approximately 3 and in the stream of supplying gas, separate and be pulled away to about 52 millimeters the staple fibre bundle of typical length, then usually help deposit on forming screen vacuumizing.The fiber of random deposition is then together adhered to one another.The instruction example of airlay method comprises the United States Patent (USP) the 4th of Laursen etc., 640, the United States Patent (USP) the 4th of the DanWeb method of having described in No. 810 (having transferred Scan Web ofNorth America Inc), Kroyer etc., 494, the United States Patent (USP) the 5th of No. 278 and Soerensen, 527, the United States Patent (USP) the 4th of the Kroyer method of having described in No. 171 (having transferred NiroSeparation a/s), Appel etc., the method of 375, No. 448 (having transferred Kimberly-Clark Corporation) or other similar method.

Inventor's discovery, in order to the SYNTHETIC OPTICAL WHITNER on cleaning objects surface, for example chlorine bleach liquor, chlorine and sodium bisulfite may have a negative impact to the solvation chromotropic dye, can not cause colour-change even bacterium does not exist also.Therefore, another aspect of the present invention comprises that the SYNTHETIC OPTICAL WHITNER with the solvation chromotropic dye detects colorant in the cleaning piece.Indicator can for example be 2,2 ', 5,5 '-tetramethyl benzidine, it is colourless under normal circumstances, look reddens when being exposed to chlorine or clorox.Indicator is the combination of starch and iodine also, and it turns black in the presence of chlorine or hypochlorite.Also another kind of indicator magenta can be used for detecting sulphite, such as Sodium Pyrosulfite.Pinkish red pinkiness becomes colourless when being exposed to sulphite.Like this, can be sensitive to bacterium with some Region specification of cleaning piece, other Region specification is sensitive to SYNTHETIC OPTICAL WHITNER and sanitas, can produce the colour-change combination so that contain the surface of active bleaching agent, and the user bacterial contamination and SYNTHETIC OPTICAL WHITNER can be made a distinction.The SYNTHETIC OPTICAL WHITNER indicator can be printed as " SYNTHETIC OPTICAL WHITNER " literal spelling pattern that is hidden on the cleaning piece, so that as long as with the cleaning piece SYNTHETIC OPTICAL WHITNER of nuzzling up, literal " SYNTHETIC OPTICAL WHITNER " will manifest, and the simultaneous SYNTHETIC OPTICAL WHITNER may cause any other colour-change that the solvation chromotropic dye occurs.The amount of SYNTHETIC OPTICAL WHITNER indicator only need be enough to cause the colour-change that can be discovered by naked eyes, and is identical with the quantitative range of solvation chromotropic dye.

The inventor also thinks, also a small amount of sample of for example following material might be included on the bar (indicating strip): a) the solvation chromotropic dye of bacterial detection; B) chlorine/hypochlorite test material is such as tetramethyl benzidine; C) oxygenant detection agent is such as the mixture of starch and potassiumiodide; D) hydrosulphite indicator such as magenta; E) nitrite detection reagent.Like this, can there be multiple quality indicator to provide for example state or the quality of food.

In another aspect of the present invention, can use coating at base material, detect dyestuff generation crystallization to stop, thereby obtain that microorganism is had more highly sensitive coating.It is desirable to, the coating that has the homogencous dyes molecule on the surface can have higher sensitivity to microorganism.Each dye molecule can freely interact with the microorganism cells film.On the contrary, the dyestuff small-crystalline at first must dissolve, then could permeates cell membranes.Although do not want to be bound by theory, but we think that still hydroxypropyl-beta-cyclodextrin, hydroxyethyl-β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-γ-cyclodextrin, hydroxyethyl-γ-cyclodextrin (hereinafter are referred to as " cyclodextrin ", all available from Cerestar International of Hammond, IN, USA) can hinder the crystallization of dyestuff, make to be manifested more bright-coloured dye colour on the base material.Find the significant quantity of cyclodextrin between the 0.001-2 weight percent, between the suitable 0.01-1 weight percent, also preferably between the 0.025-0.5 weight percent.

Find that also some tensio-active agent can help dyestuff to detect microorganism.Suitable tensio-active agent is nonionic surface active agent especially, such as ethoxylated alkylphenol, ethoxylation and propoxylated fatty alcohol, ethylene oxide-propylene oxide block copolymer, (C ₈-C ₁₈) ethoxylation ester, oxyethane and the long-chain amine of lipid acid or condensation product, oxyethane and the alcohol of acid amides, the condensation product of alkyne diol and their mixture.Each specific examples of suitable nonionic surface active agent includes but not limited to methyl glucose polyethers-10, PEG-20 methyl glucoside SUNSOFT Q-182S, PEG-20 Tego Care PS, C _11-15Pareth-20, ceteth-8, ceteth-12, dodoxynol-12, laureth-15, PEG-20 Viscotrol C, polysorbate20, steareth-20, polyoxyethylene-10 cetyl ether, polyoxyethylene-10 octadecyl ether, polyoxyethylene-20 cetyl ether, polyoxyethylene-10 oleyl ether, polyoxyethylene-20 oleyl ether, ethoxylized nonylphenol, ethoxylation octyl phenol, ethoxylation 4-dodecylphenol or ethoxylation (C ₆-C ₂₂) Fatty Alcohol(C12-C14 and C12-C18) (comprising 3-20 ethylene oxide moiety), polyoxyethylene-20 isocetyl ether, polyoxyethylene-23 glycerol laurate, polyoxyethylene-20 glycerine the moon stearate, the PPG-10 methyl glucose ether, the PPG-20 methyl glucose ether, polyoxyethylene-20 sorbitan monoesters, polyoxyethylene-80 Viscotrol C, polyoxyethylene-15 tridecyl ether, polyoxyethylene-6 tridecyl ether, laureth-2, laureth-3, laureth-4, the PEG-3 Viscotrol C, PEG 600 dioleates, PEG 400 dioleates and their mixture.Commercially available nonionic surface active agent can comprise the ofAllentown available from Air Products and Chemicals, Pennsylvania's Series alkyne diol tensio-active agent and available from Fischer Scientific of Pittsburgh, Pennsylvania's The series polyoxyethylene surfactant.

Also can adopt tackiness agent, to promote colorant fixing on base material.For example, can adopt water-soluble organic polymer as tackiness agent.The water-soluble organic polymer that one class is suitable comprises polysaccharide and derivative thereof.Polysaccharide is the polymkeric substance that contains carbohydrate repeating unit, and repeating unit can be cationic, anionic, non-ionic type and/or amphoteric ion type repeating unit.In a specific embodiment, polysaccharide is non-ionic type, cationic, anionic and/or amphoteric ion type ether of cellulose.Suitable non-ionic celluloses ether can include but not limited to alkyl cellulose ether such as methylcellulose gum and ethyl cellulose; Hydroxy alkyl cellulose ether such as Natvosol, hydroxypropylcellulose, hydroxypropyl are with butyl cellulose, hydroxyethyl hydroxypropylcellulose, hydroxyethyl hydroxybutyl cellulose and hydroxyethyl hydroxypropyl hydroxybutyl cellulose; Alkyl hydroxy alkyl cellulose ether such as methyl hydroxyethylcellulose, methylhydroxypropylcellulose, Type 3U, ethyl hydroxypropyl cellulose, methyl ethyl hydroxyethyl cellulose and methyl ethyl hydroxypropyl cellulose etc.

According to another aspect of the invention, find that dyestuff also can provide the quantity information of its microorganism that exposes.For example, the Reichardt dyestuff shows strong negative solvation metachromatism.Be that the Reichardt dyestuff can occur from blueness to colourless colour-change in the presence of one or more microorganisms.But the degree visual inspection of dyestuff variable color or use Instrument measuring is to provide sxemiquantitative and/or the quantitative correlation with microorganism concn.For example, the color of the color of the test dyestuff that reacted and contrast dye can be compared (for example range estimation or by instrument), contrast dye by aspect the responsiveness of microorganism with the same or similar compound formation of test dyestuff.Also can adopt multiple contrast dye, they are corresponding to different microorganism concns.For example, can adopt five kinds of contrast dyes, they respectively with every milliliter 10 ³, 10 ⁴, 10 ⁵, 10 ⁶With 10 ⁷The microorganism concn of individual colony-forming unit (CFU) reacts.When comparing, can select one or more colors to be same as or substantially be similar to contrast dye with the test dyestuff of test sample reaction.Then can by selected contrast dye and corresponding known microorganisms concentration, determine the central microorganism concn (or concentration range) of test sample.Thereby can obtain quantitatively (being concrete concentration) or sxemiquantitative (concentration range) result with this technology.

If needed, can measure the colour intensity of dyestuff, with the actual quantity of one or more microorganisms of existing in the confirmed test sample better.In one embodiment, measure colour intensity with the optics reader.The actual configuration of optical readings device and structure generally have multiple, and those skilled in the art easily understand.Usually, the optical readings utensil have can electromagnetic radiation-emitting light source and detector that can recording signal (for example transmitted light or reflected light).Light source can be any device that electromagnetic radiation can be provided well known in the art, the light of described electromagnetic radiation such as visible or nearly visual range (for example infrared light or UV-light).For example, can be used for suitable light source of the present invention and include but not limited to photodiode (LED), photoflash lamp, cold-cathode fluorescence lamp, electroluminescence lamp etc.Illumination can be multiplex transmission and/or collimation transmission.In some cases, pulse is carried out in illumination, to reduce any background interference.In addition, illumination can be continuous, perhaps can be with continuous wave (CW) and pulsing light combination, wherein a plurality of illuminating bundle multiplex transmission (for example pulsed light beam and CW light beam multiplex transmission) are so that can distinguish the signal that the signal of inducing in the CW source and impulse source are induced.For example, in some embodiments, LED (for example aluminum gallium arsenide red diode, gallium phosphide green diode, phosphatization gallium arsenide green diode or InGaN purple/blue/UV (UV) diode) is used as pulse illumination source.A commercial examples that is applicable to suitable UV LED excitation diode of the present invention is Model NSHU55OE (Nichia Corporation), and it launches into full width at half maximum (FWHM) with the luminous power of 750-1000 microwatt under the forward current of 10 milliamperes (3.5-3.9 volts) be that 10 degree, peak wavelength are that 370-375 nanometer and spectral half width are the light beam of 12 nanometers.

In some cases, light source can provide diffuse illumination to dyestuff.For example, can adopt simply multi-point source array (for example LED), the illumination of relative diffusion is provided.It is electroluminescence (EL) device that another kind can provide in relatively inexpensive mode the suitable especially light source of diffuse illumination.The EL device is capacitor arrangement normally, and it has utilized the luminescent material (for example phosphorus particle) that sandwiches between the electrode, and at least one electrode is transparent, to allow light penetrate.Apply voltage between electrode, the electric field that will change in the middle of luminescent material causes the luminescent material emission of light.

Detector can be any device that can sensing signal well known in the art usually.For example, detector can be the electronic imaging detector that is configured to carry out spatial discrimination.Some examples of this electronic imaging sensing detection device comprise high-speed linear charge coupled device (CCD), CID (CID), CMOS (Complementary Metal Oxide Semiconductor) (CMOS) device etc.This imaging detector is the two-dimensional array of electronic light sensors normally for example, but also can use the linear imaging detector (for example linear CCD detector) that comprises single wire probe pixel or optical sensor, for example is used for the detector of scan image.Each array comprises the known unique location of a cover, can be referred to as " address ".Each address in the visual detector is occupied by the sensor that covers certain area (for example usually being shaped to the zone of square or rectangular).This zone is commonly called " pixel " or pixel region.The detector pixel for example can be CCD, CID or cmos sensor, and perhaps any other can detect or measure device or the sensor of light.Each size that detects pixel is can be greatly different, and diameter or length can be low to moderate 0.2 micron in some cases.

In other embodiments, detector can be the optical sensor that lacks spatial discrimination.For example, the example of this optical sensor can comprise photomultiplier transit device, photorectifier such as avalanche photodide or silicon photoelectric diode etc.Silicon photoelectric diode is sometimes more favourable, because they are not only inexpensive but also sensitive, can high-speed cruising (rise time is short/and bandwidth is high), be easily integrated in most other semiconductor technology and the monolithic integrated circuit.In addition, the silicon photoelectric diode body is small, and this can make them be readily integrated in various types of detection systems.If the use silicon photoelectric diode, the wavelength region that transmits so can in their sensitivity range, be the 400-1100 nanometer.

The optical readings device can adopt any known detection technique usually, comprises such as luminous (such as fluorescence, phosphorescence etc.), absorption (such as fluorescent absorption or non-fluorescent absorption), diffraction etc.In a specific embodiments of the present invention, the optical readings device is measured colour intensity as the function of absorbancy.In one embodiment, absorbance reading is with DynexTechnologies of Chantilly, and the Model#MRX trace titre plate reader of Virginia is measured.In another embodiment, absorbance reading is to measure with the routine test that is referred to as " CIELAB ", and described test is at the F.Cost work Pocket Guide to DigitalPrinting, Delmar Publishers, Albany has discussion in the 144th and 145 page of NY.ISBN 0-8273-7592-1.This method has defined three variables L ^*, a ^*And b ^*, they are corresponding to three characteristics of the aware colors of color-based perception theory of relativity (opponent theory of color perception).The implication of three variablees is as follows:

L ^*=luminance brightness (or luminous intensity), scope from 0 to 100, wherein 0=is dark, the 100=light;

a ^*=red/green axle, approximately from-100 to 100; On the occasion of being red, negative value is green;

b ^*=Huang/blue axle, approximately from-100 to 100; On the occasion of being yellow, negative value is blue.

Because the CIELAB color space has the vision homogeneity to a certain extent, can calculate single numerical value, the difference between two kinds of colors that this numerical value representative perceives.This difference is called Δ E, by to three difference (Δ L between two kinds of colors ^*, Δ a ^*With Δ b ^*) the sum of squares root of making even calculate.In the CIELAB color space, each Δ E unit approximates greatly " just perceptible " difference between two kinds of colors.Therefore CIELAB is the good measure that does not objectively rely on the colour system of apparatus, can be used as the purpose that the reference color space is used for color management and colour-change expression.Use this test method(s), colour intensity (L ^*, a ^*And b ^*) just can for example measure with the hand spectrophotometer (model C M2600d) of Osaka, Japan Minolta Co.Ltd..This instrument adopts the D/8 geometrical shape that meets CIE No.15, ISO 7724/1, ASTME1164 and JIS Z8722-1982 (diffuse illumination/8 degree angle viewing systems).Sample surfaces is accepted by the sample measurement optical system with the D65 light of the angle reflection that departs from this normal to a surface 8 degree.Another suitable optical readings device is the reflectance spectrophotomete of describing in No. the 2003/0119202nd, the U.S. Patent application publication of Kaylor etc., for all purposes with described patent application by reference integral body be attached to herein.Equally, also the transmission-type detection system can be used for the present invention.

No matter colour intensity with which kind of mode is measured, result and predetermined detection curve can be compared in some embodiments.Detection curve is to produce by the dye strength under the various known microorganisms concentration is drawn.Like this, just can measure the color of the test dyestuff that reacts, and easily with detection curve it is associated with microorganism concn, to provide to the user quantitatively or semi-quantitative results.Although can be for widely microorganism making detection curve, also imagination can be made detection curve for the microorganism of single type.Therefore, colour intensity can be associated with detection curve for the purpose microorganism of application-specific.For example, can select the dyestuff that shows colibacillary particular reactive.In case the generation colour-change then just can be with intensity and the predetermined E. coli detection curvilinear correlation connection of color.In addition, also can make a plurality of detection curves for polytype microorganism.

Correlating method correlating method described above can be carried out automatically and/or by hand.For example, can choose wantonly and adopt microprocessor automatically to select required corresponding technology, and the observed value that will get self-detector converts the result of quantitative or sxemiquantitative indicator microoraganism concentration to.Microprocessor can comprise storage capacity, to allow the user can recall last several result.Those of skill in the art will recognize that and to use any suitable computer readable storage devices, such as RAM, ROM, EPROM, EEPROM, flash card, digital video light, Bernoulli magnetic tape machine etc.If needed, available liquid-crystal display (LCD) or LED demonstration sends the result to user.

Above-mentioned corresponding technology can be carried out with various ways according to the present invention.For example, can adopt the base material with detection zone, detection zone provides any amount of independent detection zone (such as lines, point etc.), and this is so that user's concentration of one or more microorganisms in the middle of the determination test sample better.Identical test dyestuff can be contained in each zone, perhaps can contain different dyestuffs with dissimilar microbial reaction.Such as some dyestuffs are more sensitive to gram positive bacterium, and some dyestuffs are then more sensitive to gram negative bacterium.Like this, just can detect more than one type microorganism.Also can carry out selective control to the test dye strength, so that the detection sensitivity of desired level to be provided.For example, when suspecting that the microorganism level is low, can provide the detection sensitivity of higher level with higher concentration.If needed, base material also can have the check plot that has applied contrast dye, and contrast dye is same or similar with the test dyestuff.The check plot is usually nondiscoloration in process of the test, and it can be used for carrying out quantitatively and/or the sxemiquantitative comparison like this.Similar to detection zone, the check plot also can provide any amount of isolated area.For example, the check plot can have the zone corresponding to different predetermined microorganism concns (for example aforesaid concentration).In addition, the dyestuff that dissimilar microorganism is had the different sensitivity level can be contained in described zone.

Base material can be formed by any various materials that can apply dyestuff.For example, base material can be formed by film, paper, non-woven, knitted fabrics, woven cloths, foam etc.In a specific embodiment, base material is to be generally used for the facial tissue material that label is made, such as paper, polyester, polyethylene, polypropylene, polybutene, polymeric amide etc.Tackiness agent such as pressure-sensitive adhesive, hot activation tackiness agent, hot melt adhesive etc. can be applied on one or more surfaces of facial tissue material, it be adhered on the required article helping.The suitable example of pressure-sensitive adhesive comprises for example acrylic tackiness agent and elastomer precursor gum stick.In one embodiment, pressure-sensitive adhesive is based on the multipolymer of acrylate (for example 2-EHA) and polar comonomers (for example vinylformic acid).The thickness of tackiness agent can be approximately 0.1 to the about scope of 2 mils (2.5-50 micron).Also can adopt the barrier liner that before tackiness agent uses, is in contact with it.Barrier liner can comprise and well known to a person skilled in the art any various material, such as silicone paint paper or film substrate.In use, base material and the tackiness agent that is subject to processing stripped down from barrier liner.Subsequently, tackiness agent is placed on the desired position neighbour, so that the base material that is subject to processing is exposed to environment.

With reference to Figure 17 another embodiment of the invention is described, wherein base material is lateral flow device 20.In particular, device 20 has the porous-film 23 that serves as fluid medium, and it is chosen wantonly and is being supported by the rigid material (not shown).In general, porous-film 23 can be made by any various materials wherein by test sample.For example, can include but not limited to the natural materials of natural materials, synthetic materials or process synthetic modification in order to the material that forms porous-film 23, such as polysaccharide (for example cellulose materials such as paper and derivatived cellulose such as rhodia and Nitrocellulose); Polyethersulfone; Polyethylene; Nylon; Polyvinylidene difluoride (PVDF) (PVDF); Polyester; Polypropylene; Silicon-dioxide; Inorganic materials such as inactivation aluminum oxide, diatomite, MgSO ₄, or other inorganic fine material, described inorganic materials is evenly distributed in the porous polymer matrix, and described polymkeric substance is vinylchlorid, VCP and vinyl chloride vinyl acetate copolymer; Cloth, (for example cotton) of natural appearance and synthetic (for example nylon or artificial silk); Porous gel such as silica gel, agarose, dextran and gelatin; Polymeric membrane such as polyacrylamide etc.In a specific embodiment, porous-film 23 is formed by Nitrocellulose and/or polyether sulfone materials.It should be understood that term " Nitrocellulose " refers to cellulosic nitric ether, can only be Nitrocellulose, perhaps also can be the mixed ester of nitric acid and other acid aliphatic carboxylic acid of 1-7 carbon atom (as have).Device 20 also can have absorption pad 28.Absorption pad 28 usually receives and has moved by the fluid of whole porous-film 23.Known in this field, absorption pad 28 can help to promote to pass capillary action and the fluid flow of film 23.

The user can directly be applied to test sample on the part of porous-film 23 when the microorganism that begins to detect in the test sample, and then test sample is with regard to removable this part of passing through.Perhaps, can at first test sample be applied to sampling pad (not shown) and/or the cooperation pad (not shown) that is communicated with 23 one-tenth fluids of porous-film.Can be used for forming the sampling pad and cooperate some suitable materials of pad to include but not limited to Nitrocellulose, Mierocrystalline cellulose, porous polyethylene pad and glass fiber filter paper.No matter where test sample is applied to, it all can move to the detection zone 31 that is defined by porous-film 23, and detection zone can send the existence that signal shows microorganism.Specifically, as shown in figure 17, detection zone 31 comprises when contacting with one or more microorganisms and to show the test dyestuff that can detect colour-change.Determinator 20 has also adopted check plot 32, and it is applied with contrast dye, the optional downstream that is placed on detection zone 31.Check plot 20 is usually nondiscoloration in process of the test, so it can be used for sxemiquantitative and/or quantitative comparison.

That sometimes tests dyestuff and contrast dye applies mode so that they can not diffuse through the matrix of porous-film 23 basically.This is so that the user can easily detect the color of dyestuff after the required reaction times.For example, dyestuff can form ionic linkage and/or covalent linkage with the functional group that exists on the surface of porous-film 23, and they just fixedly remain on the film like this.In one embodiment, positively charged dyestuff can form ionic linkage with the electronegative carboxylic group of existence on some porous-films (for example Nitrocellulose) surface.Perhaps, some can be stoped substantially dyestuff to join in the dye solution to the composition of the matrix diffusion of porous-film 23.In other cases, may not need immobilization, opposite dyestuff can be diffused in the matrix of porous-film 23 and react with test sample.

Following examples help explanation each embodiment of the present invention.

The embodiment material

Unless otherwise, all reagent and solvent be all available from Aldrich ChemicalCompany Inc. (Milwaukee, WI), and use without being further purified namely.The microorganism of using in the research is:

1. Gram-negative (work)

Intestinal bacteria (Escherichia coli) (ATCC#8739)

Pseudomonas aeruginosa (Psuedomonas aeruginosa) (ATCC#9027)

Salmonella choleraesuis (Salmonella choleraesuis)

Gardnerella vaginalis (Gardnerella vaginalis)

2. Gram-positive (work)

Streptococcus aureus (Staphylococcus aureus) (ATCC#6538)

Staphylococcus xylosus (S.Xylosis)

Lactobacterium acidophilum (Lactobacillus acidophilus)

3. Gram-positive (extremely)

Streptococcus aureus (Staphylococcus aureus) (ATCC#6538)

Staphylococcus xylosus (S.Xylosis)

4. yeast (work)

Candida albicans (Candida Albicans)

5. mould (work)

Aspergillus niger (Aspergillus Niger)

6. viral

-1 type poliovirus

-herpes simplex types 1 virus (HSV-1)

-rhinovirus

-Measles virus

-vaccinia virus

-A type influenza virus

All viruses are all available from the GibraltarLaboratories in N.J. Fairfield city, Inc..Reichardt dyestuff (phenolic acid 2,6-phenylbenzene-4-(2,4,6-triphenyl-1-pyridine

)) and bromination 1-docosyl-4-(4-Vinyl phenol base)-pyridine

Aldrich Chemical Company available from Wisconsin, USA Milwaukee city.Other part cyanines that this institute uses are synthetic in company, hereinafter describe in detail.

Synthesizing of part cyanines class material

Bromination 1-docosyl-4-(4-Vinyl phenol base)-pyridine

Commercially available available from AldrichChemical Co. (Milwaukee, WI), directly use.

The other example of merocyanine dye is synthetic at the use for laboratory two-step reaction.

Show in Fig. 3 with the synthetic dyestuff for preparing of synthetic method.

As shown in Figure 4, methyl-iodide is slowly joined in the 50ml aqueous isopropanol of the δ-picoline under stirring in the ice bath.Add complete after, reaction is heated to reflux and continue refluxed 2 hours.Then cooling solution in ice bath goes out sedimentation and filtration, washs with cold alcohol in Büchner funnel.Then with powder in stink cupboard dry 2 hours.The yield of crude product is 18.6 grams.Crude product is not further purified, and is directly used in next step.

As shown in Figure 5, N-methyl-δ-picoline (9.4g, 0.04 mole) and vanillin food grade,1000.000000ine mesh (6.1g, 0.04 mole) under agitation all are dissolved in the 50ml ethanol.Add piperidines (3.4g, 0.04mole) to this solution, the gained mixture refluxed 16 hours.Then reaction mixture is cooled off in ice bath, leach product with Büchner funnel, use cold washing with alcohol.

Then the 250ml 0.2 volumetric molar concentration potassium hydroxide solution with the thick dyestuff (wherein R=methyl) of said structure 13 stirred 60 minutes, formed zwitter-ion, leached with Büchner funnel.Then with dyestuff crystallization from 1: 1 water/carbinol mixture of minimum quantity.Yield is 9.4g (98%).

Other dyestuff synthesizes by similar mode from corresponding alkyl iodide.Following table 1 shows compound and the yield of the dye structure 13 of three kinds of different R groups that obtain.

The alkyl derivative that table 1. synthesized and the yield that obtains

Alkyl	Yield
		The R=methyl	98
The R=hexyl	92
		The R=dodecyl	87

Embodiment 1:Reichardt dyestuff (in the acetonitrile solvent) is applied to the surface of polluting in advance

For studying these dyestuffs as the purposes of spray, the solution (160mg is in the 10mL acetonitrile) of preparation Reichardt dyestuff.Utilization produces spray system with the spray bottle of aerosol propellants.

The live chickens leg was at room temperature displayed several days, to guarantee high bacteria levels.As shown in Figure 6, chicken leg is placed on the surface of ceramic plate and removes (6A) after several seconds, then this surface is blotted to remove any chicken extract vestige (6B).Then, with spray bottle the Reichardt dye solution is sprayed onto (6C) on this surface.To prove conclusively repeatability, drawn similar result with another piece chicken revision test.

After being sprayed onto indicating dye solution on this surface, the zone of whole contacted chicken decolouring (that is to say, the color of indication spray becomes very dim or colourless from blueness), the profile (6D) of generation chicken.Dyestuff is let slip chicken on this surface accurate location is decoloured fast.

Embodiment 2:Reichardt dyestuff (in the Virahol) is applied to the surface of pollution

Virahol as carrier is studied, and Virahol may have other benefit because of its disinfecting power.The Reichardt dyestuff is dissolved in (the 160mg dyestuff is dissolved in the 10mL Virahol) in the Virahol.As " reality " surface, test the bacterial contamination situation on it with the plastic door handle.Stain the surface of one of them door handle with the juice of displaying chicken.It is not comtaminated that other door handle keeps, to compare.To all in addition wipings of two kinds of door handles.The aqueous isopropanol of dyestuff is sprayed on two kinds of surfaces.Decoloured into colourlessly by the Reichardt dyestuff from blueness, just easily observe out the Polluted area of door handle.

Embodiment 3: the false positive of Reichardt dyestuff on contaminated surface in the test of indication spray in the Virahol

Confirmed that the Reichardt dye indicator has very high sensitivity to the bacterium from chicken.Be the false positive situation of test indicator, used other composition such as lipid and the protein of chicken extract.Adopted chicken soup, purpose is the height possibility of utilizing its abacterial characteristic and containing the potential interference material.

With in the can of just having opened

Chicken soup (Campbell Soup Co., NJ can be from the commercially available acquisition in retail grocery store), is used to ceramic surface with pipette, extract

Paper handkerchief is dried.Also with the juice of ageing chicken with pipette, extract to the different positions of ceramic surface, dry, as known positive control.Reichardt dye indicator (160mg is in the 10ml Virahol) is sprayed on the ceramic surface, knows and see that only containing the one side of displaying chicken extract (and therefore containing bacterium) is decoloured.Can reach a conclusion from this experiment, in the situation that chicken, really bacterium caused the decolouring response, rather than some secondary compositions such as chicken fat or protein.

Embodiment 4:Reichardt dyestuff indication spray is as the cleaning additive on the contaminated surface

Also tested Reichardt dyestuff purposes as cleaning additive in the Virahol spray.As shown in Figure 7, will display chicken extract and vertically be applied on the left and right sides two halves of square ceramic surface (7A).A one side of something is except using

Paper handkerchief outside the wipe surfaces, does not clean energetically.Half of for B, then Kimberly-Clark Professional Moisturizing Instant HandAntiseptic (60% ethanolic soln, Roswell GA) is applied on the towel, in order to clean this surface (7B).Then apply Reichardt dyestuff spray, to determine cleaning whether create a difference (7C).The clear discovery, although sanitising agent institute is helpful, but some zone " is missed " (7D).Then, the base portion halves that again cleans energetically two fringe area with K-C Professional Antiseptic is divided.When spraying these zones (7F), do not decolour, this has confirmed surperficial cleaned (7G) again.

The decolouring of the paper material of embodiment 5:Reichardt dyestuff coating

The ability of the top coat response bacterial contamination of this experiment test Reichardt dyestuff.As shown in Figure 8, with Reichardt dye solution (80mg/10mL acetonitrile) brushing a piece of paper.Add 10 to this paper ⁷, 10 ⁶, 10 ⁵With 10 ⁴100 μ L aliquots containigs (8B) of CFU/mL intestinal bacteria or streptococcus aureus solution.Water is as negative control.When by this bacterial contamination of two types, dye colour is eliminated fast, faster for streptococcus aureus (8D).Determine afterwards, although two kinds of bacterial solutions really 10 ⁷CFU/mL concentration, but the actual concentrations of streptococcus aureus solution is 7X10 ⁷CFU/mL, intestinal bacteria solution is 1X10 by contrast ⁷CFU/mL.Compare with the viewed quick decolouring of bacterial solution (less than 1 minute), water causes just that behind several minutes dyestuff decolours a little.

A self-adhesion stickers paper (Avery-Dennison) is also used Reichardt dye solution (160mg/10mL acetonitrile, the 80mg/10mL acetonitrile) brushing of two kinds of different concns.Stickers is applied to The lid bolt mechanism of wet facial tissue case (lid and latchmechanism).Put on one's gloves 10 ⁷The CFU/mL streptococcus aureus is transferred on the surface of stickers.Although two kinds of concentration are all decoloured fast, color disappears more obviously on the lower surface of dye strength, shows to exist to provide to detect and the best coating concentration of strong visual contrast.Stickers can be multiple application and provides easily and the means of rapid detection bacterial contamination.

Embodiment 6: carry out the quantitative of bacterial concentration with the Reichardt dyestuff

By with the bacterium on the Reichardt dye liquid testing substrates, rather than allow and be attached to bacterium (embodiment 5) in the dyestuff contact liq on the base material, recognize the potential new purposes based on the bacterium indicator of Reichardt dyestuff.The focus of this experiment is to determine how dye solution responds the bacterium that is positioned at lip-deep concentration known.With 10 of 100 μ l ⁸CFU/ml gram positive bacterium drop On the towel (Fig. 9 A).Add a Reichardt dyestuff (160mg is in the 10ml acetonitrile) that is dissolved in acetonitrile (9B) to this spot.

For comparing drop one dyestuff spot and dry on towel, the bacterium of adding same amount.Decolour immediately once being added to bacterial spot Reichardt dyestuff.Contrast places the reaction of the bacterium on the cellulosic towel that contains dyestuff just to decolour in several minutes therewith.Add several dyestuffs (9C) to this bacterial spot, the decolouring phenomenon proceeds to the 4th again, and be purple (9D) this moment just always.Attempt to recover to be coated with dyestuff by add acetonitrile with transfer pipet

Dye colour on the towel, but fail (9E).

Embodiment 7: carry out the test of bacterium indicator titration with the Reichardt dyestuff

The bacterium that adheres to base material makes fast this discovery of decolouring of dye solution, and reaction can reach this fact of terminal point, excites us to probe into the ability that dyestuff provides bacterium CFU/ml quantitative information.The purpose of this experiment is the bacterium that adheres to base material with the various concentration of dyestuff titration, and whether the amount that is defined as the required dyestuff of stable color changes with bacterium CFU/ml.

The streptococcus aureus serial dilution suspension of each 100 μ l of drop on the paper handkerchief.Then ((40mg/10ml) is drawn on germy each spot of drop with the acetonitrile solution of several (10 μ l) Reichardt dyestuffs with transfer pipet.Dye solution also has color at the beginning, but almost at once (less than a second) just decolouring, again to several dyestuffs of same spot drop, until dyestuff no longer decolours and purple/blueness does not weaken.Repeating this operation corresponding to each spot that the different concns bacterium is arranged.

The good correlation of the bacterial contamination level on as a result demonstration and surface or the base material.

Embodiment 8: the bacterium titration of elderly woman urine

For the practical use of this novel method is described, the elderly woman urine sampling product that will compile (100 μ l) point drops on the cellulosic towel, produces several spots, and each spot has the urine of 100 μ l volumes.Two kinds of dye solutions have been used in the titration research: 40mg dyestuff/10ml acetonitrile and 160mg dyestuff/10ml acetonitrile.Then dye solution is dripped on the urine spot with 10 μ l aliquots containig points, the continuation point drops to blueness/purple dye color and is kept (be about to dyestuff and join urine, until color continues to keep).Table 2 has provided every kind of dye solution for making dye colour keep stable (i.e. no longer decolouring) required volume.Known elderly woman urine has high bacterial contamination level, and this preliminary study confirms the high contamination levels in this concrete sample.

Table 2: the bacteria quantified of women's urine of compiling

Sample	The 4mg/ml dye solution	The 16mg/ml dye solution
			Urine	120μL	30μL

Dye solution that it should be noted that rarer (four times of dilutions) of use is four times of dye solution of denseer (dense four times).Like this, so that the amount that reaches capacity required is minimum, can allow indication mechanism adapt to different CFU levels seen in the different industries (food, health care etc.) by using maximum dye strength.For example, decide with storage conditions between apparent time, chicken nugget no matter where all can produce 10 ²-10 ⁹Bacteria levels.But for the worry to diseases, making food person and processor may only be concerned about 10 ⁷And above bacteria levels.On the other hand, hospital's general therapeutic because of disease, sufferer or operation may be the patient of immunocompromised host to a certain extent.Therefore, the corpsman,hospital may be concerned about much lower bacteria levels than other industry of great majority, can benefit from potentially the indicating dye concentration of the specific needs that adapts to them, in order to reduce susceptible patient's infection risk.

Embodiment 9: with the multiple-microorganism test bacterium indicator that comprises bacterium, Molds and yeasts

Press the same way as that preamble is described, the cellulosic towel as base material, is used pipette, extract bacterium and other microorganism thereon.With transfer pipet with 10 ⁷The streptococcus aureus of CFU/ml, Candida albicans (yeast), gardnerella vaginalis, intestinal bacteria, Pseudomonas aeruginosa and Lactobacterium acidophilum are drawn to (each 100 μ l) on the towel.In addition, also with transfer pipet with 10 ⁵Aspergillus niger (a kind of common mould) be drawn on the towel.Then Reichardt dye solution (160mg is in the 10ml acetonitrile) is joined each spot with 10 μ l aliquots containigs, counting causes the number that lasting color is required.

Table 3 provides for every kind of microorganism as keeping continuing the amount of the required dyestuff of purple.Observing the strongest reaction with Lactobacterium acidophilum, secondly is streptococcus aureus, gardnerella vaginalis, intestinal bacteria, Pseudomonas aeruginosa, Candida albicans, is aspergillus niger at last.Although seem that the Gram-positive streptococcus aureus is the same with the reaction of Gram-negative gardnerella vaginalis strong, for various types of bacteriums and pathogenic agent, be different for reaching the required quantity of homeostatic reaction.

Table 3: with the titration of Reichardt dyestuff to various microorganisms

Embodiment 10. usefulness bacteria cell wall component testing Reichardt dye indicators

Utilization is common in the molecule of bacteria cell wall, can know this indicator technologies clearly and how to work.Have certain general character although consist of the compound on the surface of gram positive bacterium and gram negative bacterium, their arrangement and chemical constitution are different.The adventitia of gram negative bacterium is being coated with lipopolysaccharides (LPS).LPS brings net negative charge to the surface of gram negative bacterium, helps its pathogenesis.Gram positive bacterium is being coated with in other words murein platy layer of thick peptidoglycan.This lamella is to be formed by the N-acetyl-glucosamine and the-acetylmuramic acid molecule that alternately link to each other.Also have teichoic acid in gram positive bacterium, it can be connected to-acetylmuramic acid.Although gram negative bacterium also has peptidoglycan, it is much thick that the peptidoglycan layer on the gram positive bacterium is wanted.In addition, the peptidoglycan layer of gram negative bacterium be positioned at the LPS layer below, this so that it be difficult for approaching from the surface.

The detoxification lipopolysaccharides (removing the lipid A composition) in intestinal bacteria source, the lipoteichoicacid in streptococcus faecium (Streptococcus faecalis) source, the lipopolysaccharides in intestinal bacteria source and the solution point of teichoic acid are dripped to

On the paper handkerchief.Except pure LPS, all solution all is prepared into 5% (wt/wt), 1% (wt/wt) and 0.2% (wt/wt) concentration.For safety, LPS is prepared into 0.1% (wt/wt), 0.02% (wt/wt) and 0.004% (wt/wt) concentration.Then Reichardt dyestuff (160mg is in the 10ml acetonitrile) is joined each spot with 10 μ l aliquots containigs, record produces the required dye quantity of lasting color.The experiment of reversing simultaneously is about to the cell wall constituent point and drips on the dyestuff spot on the paper handkerchief.

Teichoic acid produces the strongest reaction, all causes almost at once decolouring of dyestuff in two kinds of experiment situations.As if other compound also finally causes dye decolored really, but reaction is strong not as teichoic acid.Because the concentration that teichoic acid exists in gram positive bacterium is higher, these results have proved that not only this dyestuff provides the potentiality of CFU/mL data, and have proved the potentiality of distinguishing Gram-positive and gram negative bacterium according to intensity and the speed of reaction.

Embodiment 11: the test of chicken Related Component

Confirmed that the Reichardt dye indicator has highly sensitive to the microorganism on the live chickens meat that grows at room temperature preservation.But, consider to have false-positive potential possibility, be necessary to test indicator to the response condition of other composition such as lipid and the protein of chicken extract.To contain the canned chicken soup of chicken derived products such as lipid, protein etc. with comparing, with the caused potential interference of the material of verifying these natural appearance.

With transfer pipet with firm can opening

Chicken soup is drawn on the electric bakeware surface, uses

Towel.Also will be drawn on the electric bakeware from the juice of the live chickens meat of preservation a couple of days under the normal temperature with transfer pipet and dry, as positive control.With Reichardt dye indicator (160mg is in the 10ml Virahol) spray from the teeth outwards, clear see only containing display decolouring beyond the chicken extract (thereby containing bacterium).Can reach a conclusion from this test, in the situation that chicken, the existence of really microorganism caused the decolouring response, rather than some other composition such as chicken fat or protein.

Embodiment 12: the impact that highly basic is dye decolored on Reichardt

The interactional PRELIMINARY RESULTS of Reichardt dyestuff and cell wall constituent such as teichoic acid, and be intended to identify that potential false-positive work, prompting may be the decolourings that has inspired the Reichardt dyestuff with the reaction of acid generation.This causes this supposition, i.e. acid-alkali reaction may play a role in viewed colour-change.Therefore plan tests to test highly basic to the impact of the Reichardt dyestuff of decolouring.

Arrive with a pipette, extract number Reichardt dyestuff (160mg is in the 10ml acetonitrile)

On the towel, allow its exsiccation.Notified the compound (acetic acid and Aldrich pH of buffer 2.0) that causes colour-change with two kinds and dropped in respectively on two in these spots, this causes dyestuff to decolour fast.Respectively draw a 1N NaOH to each spot with transfer pipet, cause fast more painted (re-colorization).The blueness of Reichardt dyestuff/purple recovers behind the adding 1N NaOH.

Carry out second experiment with the indication spray, to prove conclusively these results.To display live chickens gravy with transfer pipet and be drawn on the electric bakeware surface, the style of drop will be easy to identification.The surface is blotted, spray Reichardt dyestuff indication spray (160mg is in the 10ml acetonitrile), cause dye decoloredly, surface topography is the style of chicken extract.Then a 1N NaOH point is dropped in the zone of decolouring, cause this speckle painted again.This operation is also repeated in another zone.

For the checking possibility is that 1N NaOH only acts on bacterium rather than acts on dyestuff, the NaOH equal proportion of displaying chicken extract and 1 volumetric molar concentration is mixed, and allow mixture leave standstill for 30 seconds.Then ooze the style of another identical (but a little bit smaller) with this mixture point.This solution also causes the quick decolouring of Reichardt dyestuff, still color restoration when adding 1N NaOH.

Embodiment 13: with stickers normal and the vaginal secretion test Reichardt dyestuff coating that bacterial vaginosis (BV) infects

In view of being widely current of bacillary vaginal infection, test to study the stickers of Reichardt coating dye to healthy (low pH, infect without bacterium), the pH positive/bacterial vaginosis (BV) is negative (infects without bacterium, but pH exceeds normally) response condition of the vaginal secretion sample of and the pH positive/BV positive (pH exceeds normally, known have bacterium infection).A stickers is coated with the Reichardt dye solution (160mg/10mL acetonitrile, 80mg/10mL acetonitrile, 40mg/10mL acetonitrile, 20mg/10mL acetonitrile) that brushes two kinds of different concns.With normal, BV is positive/stickers of each concentration of vaginal secretion sample test of the positive and BV feminine gender/pH positive of pH.

Normal vaginal secretion produces the most obvious dye decolored, presumably is because due to lactobacillus and these two factors combine of low pH.Secondly the decolouring situation of the BV positive/pH positive may be because exist a large amount of BV bacteriums.BV feminine gender/pH positive only makes the faint decolouring of dyestuff, may be because the quantity of lactobacillus is lacked than normal specimens.Three kinds of decolored states are easy to difference, point out this technology to have the diagnosis potentiality in the vaginal health field.

Embodiment 14: with Reichardt dyestuff indication spray test general surface

As everyone knows, bacterium can survive a few hours at desiccated surface, even a couple of days.Identify bacterium and other microorganism on the general surface and remind the human consumer or the workman notes the ability of pollution condition will helping cleaning and disinfection work, help to make spread of infection to minimize.

As shown in figure 10, come test microbes indication spray (10A) with old computer keyboard as model " reality " surface.The Reichardt dyestuff is dissolved in Virahol (160mg is in the 10mL Virahol), adds to based on aerocolloidal spraying plant.Then keyboard is sprayed to indicate solution (10B).

Keyboard spray is with Reichardt dyestuff indication solution cause decolouring fast in some areas of dye (10C).What is interesting is to only have some buttons or zone to show pollution, this is so that can specifically identify highly the button that stains, such as digital keyboard.Because keyboard often uses and cleaning seldom, this surface allows the people catch a glimpse of microorganism level on the real surface really.

Embodiment 15: the sensitivity that improves the bacterium indicator with tensio-active agent

The preparation Reichardt dyestuff (80mg/10mL acetonitrile) and

The solution of 80 (200 μ L) polyoxyethylene surfactant (available from Fischer Scientific, Pittsburgh, PA).Then use this solution coated with ceramic surface (Figure 11), make it air-dry.With another Reichardt dyestuff (80mg/10mL acetonitrile) solution drop of not containing tensio-active agent from the teeth outwards, allow equally its air-dry (11A).After the drying, on each the area of application one of drop known have a very high bacterial count display chicken extract (11B).Contain

The zone of 80 tensio-active agents (11C) with do not contain

The zone of tensio-active agent (11D) is compared, decolorization rate faster (＞20-30 second).In addition, add

Tensio-active agent also makes dyestuff easily remove from the surface.Add a small amount of water and can make from the surface and remove fully, do not make from lip-deep removing to the spot that does not contain tensio-active agent and become easy and add water.

Embodiment 16: the importance that solvent is selected

Performance to the Reichardt dye coatings that makes with various different solvents is assessed.The solution of preparation Reichardt dyestuff in acetonitrile, Virahol and dimethylbenzene is with these solution coatings

Jack-towel for kitchen use allows its air-dry (Figure 12 A and 12D).Drop 100 μ l streptococcus aureus aliquots containigs (12B, E) on the towel that is subject to processing, the colour-change of observation coating.Only based on the coating of acetonitrile solution decolour fast at drop bacterial suspension place (12C).Observe Reichardt dyestuff color even when being dissolved in acetonitrile.Other two kinds of solvent coating are not observed visible colour-change (12F).

The inventor finds, can be adjusted the concentration of Reichardt dyestuff, so that Virahol can be used as the solvent of dyestuff.So strong when although the color of dyestuff is not as acetonitrile at this moment, but easily observe fast the decolouring that responds microbial contamination and occur.

Embodiment 17:N-docosyl merocyanine dye is coated on the cotton fabric

Half new freshly-slaughtered poultry (available from the supermarket), three weeks of at room temperature preservation of transparent film will be covered with on the polystyrene dish.Collect the light yellow juice that compiles in the polystyrene dish with transfer pipet, be used for test.

Bromination 1-docosyl-4-(4-Vinyl phenol base) pyridine with 47mg

(available from Aldrich Chemical) mixes with the dimethyl formamide of 10g.A small amount of solid substance residue is arranged after the jolting, allow it precipitate.Orange supernatant liquor point is dripped on the cotton fabric of quantitatively (29.2cmx20.3cm=6.888g), form orange-yellow circle.A 1N sodium hydroxide solution is joined an orange-yellow spot on the cotton fabric, make color from orange-yellow become pink orange.

Drip on the orange-yellow spot on the cotton fabric displaying the chicken extract point, make color become very shallow yellow.Colour-change is very fast on cotton fabric.Equally, drip to pink orange areas (dyestuff+NaOH solution) on the cotton fabric with displaying the chicken extract point, the colour-change that causes is similar, namely from the pink orange very shallow yellow that becomes.

Embodiment 18:N-methyl part cyanines are coated in paper handkerchief for kitchen use and display on the urine

Collect women's urine, preservation 8 days under the room temperature in vial.The N-methyl merocyanine dye that synthesizes as mentioned above following structure.0.5g is dissolved in the 20ml deionized water, is coated to On the kitchen paper roll towel (by paper handkerchief is immersed solution), allow excessive solution drop fall, then allow the paper handkerchief of coating dry under envrionment conditions.With dyestuff paper handkerchief is dyed darkorange.

To display the urine point and drip on the orange paper handkerchief, produce immediately colour-change, become light yellow from darkorange.In contrast, will display urine and filter 0.2 micron filter, degerm and other microorganism to remove.Display behind the urine filtering point and drip to and do not cause colour-change on the paper handkerchief, this prompting is that microorganism rather than other composition displayed in the urine cause colour-change.

Embodiment 19:N-methyl part cyanines are coated in paper handkerchief for kitchen use and display on the urine

Collect women's urine, 37 ℃ of lower preservations 24 hours.Women's urine of compiling estimates that bacterial load is approximately 1x10 after the preservation under these conditions ⁵CFU/ml.The N-methyl merocyanine dye that synthesizes as mentioned above following structure 33.0.5g is dissolved in the 20ml deionized water, is coated to

On the kitchen paper roll towel (by paper handkerchief is immersed solution), allow excessive solution drop fall, then allow the paper handkerchief of coating dry under envrionment conditions.With dyestuff paper handkerchief is dyed darkorange.

To display the urine point and drip on the orange paper handkerchief, produce immediately colour-change, become light yellow from darkorange.In contrast, will display urine and filter 0.2 micron filter, degerm and other microorganism to remove.Display behind the urine filtering point and drip to and do not cause colour-change on the paper handkerchief, this prompting is that microorganism rather than other composition displayed in the urine cause colour-change.

Embodiment 20:N-methyl part cyanines are coated on paper handkerchief for kitchen use and the pet bird stool

Collect ight soil by the budgerigar in the birdcage, put into the jolting of about 10ml Atlanta household tap water.The N-methyl merocyanine dye that synthesizes as mentioned above above structure 33.0.5g is dissolved in the 20ml ml deionized water, is coated to

On the kitchen paper roll towel (by paper handkerchief is immersed solution), allow excessive solution drop fall, then allow the paper handkerchief of coating dry under envrionment conditions.With dyestuff paper handkerchief is dyed darkorange (Figure 13 A).

The suspension point of several budgerigar ight soil in tap water dripped to (13B) on the coating paper towel, produce immediately colour-change in the place that adds suspension, become light yellow from darkorange.In contrast, the household tap water point of Atlanta being dripped to the different zones of paper handkerchief, although color is diluted a little by water, should the zone still be orange.

Embodiment 21 (predictive): the indicating dye that is used for coating

Under good the stirring, N-methyl part cyanines of 1 gram Vltra tears, 0.5 gram structure 33 dissolve in the mixture of 10 gram water and 10 gram Virahols.Can be coated to this solution on the polyester film and be allowed to condition under the room temperature dryly, can detect the coating flexible film that microorganism exists to produce.

Embodiment 22 (predictive): the indicating dye in the coating

N-methyl part cyanines of 1g ethyl cellulose, 0.25g structure 33 can be dissolved in the 20 gram tetrahydrofuran (THF)s.Can be coated to this solution on the polyester film and be allowed to condition under the room temperature dryly, can detect the coating flexible film that microorganism exists to produce.

Embodiment 23: the lateral flow device

Millipore Nitrocellulose HF75 film (available from Millipore Corporation ofBillerica, MA, USA) superimposition is supported on the card (available from Millipore Corp.) at the about 30 centimetres plastics of length.With hand the aqueous isopropanol of 5 weight percent Reichardt dyestuffs is drawn striped at detection zone and check plot.With film in lab oven at 37.5 ° lower dry 1 hour.Behind baking oven taking-up film card, cellulosic core backing strap (available from MilliporeCorp., catalog number (Cat.No.) CFSP203000) is attached to an end of close check plot on the film.Card is downcut in order to the other end that connects sample pad.Then card is cut into the 4mm band, forms half bar (half stick).

After producing half bar, bacterial solution is applied to the end that detects film.Capillary action is drawn onto solution and bacterium in the detection zone, can see colour-change at detection zone.The color of contrast lines keeps the same in whole process of the test.

Embodiment 24: cyclodextrin strengthens

At first will

Paper handkerchief is dipped the aqueous solution (1 restrains in 20ml) of coating hydroxypropyl-beta-cyclodextrin (available from CerestarInternational, Hammond, IN, USA), and air-dry at ambient temperature.After the coating paper handkerchief is done, process with the aqueous isopropanol (1 weight percent) of Reichardt dyestuff, make it air-dry.Dry paper handkerchief color is purple/blueness.Cyclodextrin has hindered the crystallization of dyestuff just, and dye colour is apparent on the paper handkerchief more bright-colouredly.Test with this coating paper handkerchief and gram negative bacterium (intestinal bacteria), when 100 mul aliquots samples of the substratum that will contain 10,000CFU/ml are applied to paper handkerchief, find less than just becoming colourless 5 seconds.Find that bacterial concentration is low to moderate 500CFU/ml and this decolouring phenomenon also can occur, this need to reach 15 seconds time certainly.Therefore, it is believed that dyestuff will be present on the base material with unit molecule by hindering the dyestuff crystallization, thereby dyestuff is to the sensitivity raising of bacteria levels.The inventor thinks, careful application of paint (for example cyclodextrin) on paper handkerchief will at the monomolecular coating of substrate surface generation dyestuff, will obtain maximum sensitivity.

Embodiment 25: the dry-eye disease test

Paper handkerchief with the coating of Reichardt dyestuff is tested with " doing " bacteria samples (not in solution).Picking colony from the agar culture dish that contains a series of Escherichia coli Growth cultures is made dry-eye disease and is used.This dry-eye disease obliterating is applied to pre-wetting dyestuff On the paper handkerchief.Obliterating has the zone of bacterium colony to become colourless in second at 1-5.The use-pattern of this and wet wipes is similar, and does well.

Embodiment 26: the bleaching indicator test

The mixture of Reichardt dyestuff and TMB (TMB) is coated in

On the paper handkerchief, make it air-dry.The liquid lime chloride of dilution is applied on the paper handkerchief, causes the dye decolored and TMB of Reichardt to become orange/yellow.This shows and the bleaching indicator can be combined in the bacterium indication cleaning piece.

In last test, to having Reichardt dyestuff and TMB coating

Paper handkerchief drips intestinal bacteria suspension, and makes it be exposed to bacterium.Paper handkerchief touches the zone of bacterium less than just decolouring into white dot 10 seconds.Do not observe orange/yellow generation.

Embodiment 27: the uv-visible absorption spectroscopy of part cyanines and zwitter-ion dyestuff

The Reichardt dyestuff uses without being further purified namely.The N-n-hexyl is synthetic by relevant description with N-dodecyl merocyanine dye.Used solvent obtains Aldrich Chemical, is the HPLC level.Measure dyestuff with Shimadzu UV-1601 ultraviolet-visible spectrophotometer (ShimadzuCorporation) and absorb at the long wavelength peak of 400-800nm scope, described dyestuff is dissolved in three kinds of different solvents, is contained in to adapt in the cuvette.Following table is the result that the dyestuff of the solvent on the left side and top is tested.

	Hexyl part cyanines	Dodecyl part cyanines	The Reichardt dyestuff
				Acetone	(617.5nm green)	617nm (green)	674nm (blue-greenish colour)
Methyl alcohol	514nm (orange)	522nm (orange)	509nm (redness)
				Acetonitrile	582nm (light green-blueness)	600nm (blueness)	623nm (blueness)

Merocyanine dye also shows absorption at nearly 400nm place except longer wavelength absorbs, this can change the color that perceives.

Significantly, according to the spectrophotometry result, these microorganism detection dyestuffs are in being dissolved in different solvents the time, they demonstrate the maximum wavelength peak be absorbed with large skew (＞10nm).

Embodiment 28: virus detects

Confirm that chromogen detects the ability that virus exists according to the present invention.Prepare 1 type poliovirus, herpes simplex types 1 virus (HSV-1), rhinovirus, Measles virus, vaccinia virus and A type influenza virus, and they are inoculated into the MA-104 tire monkey-kidney cells of Dulbecco ' s Modified Ealge substratum (DMEM) propagating and breeding of adding foetal calf serum to 5% concentration, at 37 ℃ ± 1 ℃, 5%CO ₂Lower incubation 6 days.(cytopathic effect CPE), becomes circle, shrinkage, cracking, pyknosis etc. as observing, the detection viral proliferation to cell disintegration situation by microscopic examination cells infected lamella (cell sheet) at least 50% cell sheets.Cytotoxicity is measured as the degree of the cell disintegration that material produces when virus-free.Carry out the titration of virus with ten times of DMEM serial dilutions, 4 of each dilutions repeat MA 104 cultures, and each repeats sample and inoculates with 0.1 milliliter of viral dilution liquid.The method of Reed and Muench of pressing is measured, and the degree of virus replication is calculated as tissue culture infective dose (TCID ₅₀).

With the stickers (160 milligrams/10 milliliters acetonitriles, 80 milligrams/10 milliliters acetonitriles, 40 milligrams/10 milliliters acetonitriles and 20 milligrams/10 milliliters acetonitriles) of Reichardt dyestuff coating as testing surface.With 50 milliliters of undiluted virus (TCID in substratum ₅₀10 ^-8Poliovirus/mL; TCID ₅₀10 ^-7HSV-1/mL; TCID ₅₀10 ^-7Rhinovirus/mL; TCID ₅₀10 ^-6Measles virus/mL; TCID ₅₀10 ^-6Vaccinia virus/mL and TCID ₅₀10 ^-7Influenza virus/mL) be added drop-wise on every stickers allows its static 3 minutes, then wipes drop away with cotton swab.For the rhinovirus and the poliovirus that are diluted in substratum and the salt solution, the aliquots containig of single culture base, virus-free cell culture medium and virus-free cell cultures salt solution as control sample, is allowed its static 3 minutes equally, then wipe away.For remaining virus (not adding dilution in its original substratum uses), only adopt the substratum contrast.

As if for poliovirus, saline control can be disturbed dyestuff, substratum does not then cause colour-change.Therefore the rest part of experiment uses the diluent of poliovirus in substratum.Should virus in substratum, make ten times of serial dilutions, and get 50 mul aliquots samples and be applied to every stickers.Allow drop after static 3 minutes, it is wiped away from stickers.For rhinovirus, find that substratum produces interference to note, saline control does not then cause dye colour to change.Therefore, the ten times serial dilutions of this virus in salt solution are applied to stickers with 50 mul aliquots samples, wipe away after 3 minutes.For poliovirus and rhinovirus, be low to moderate 10 ^-6Stickers all can variable color when (ten times of serial dilutions the 6th time dilution), and this shows that dyestuff coating stickers has these viral sensitivity of detection (decolouring is slightly strong due to the poliovirus).For HSV-1, A type influenza virus, Measles virus and vaccinia virus, only (undiluted) virus liquid dropping point with 50 microlitres drops on the stickers.To subsequently viewed decolouring situation and virus-free control medium and Salmonellas (10 ⁸CFU/mL) the viewed decolouring situation of positive control compares.Although decolouring is so strong not as the viewed decolouring of Salmonellas when being exposed to undiluted HSV-1 virus, the stickers decolouring that causes is stronger than rhinovirus and the viewed decolouring of poliovirus.Response A type influenza virus, vaccinia virus and Measles virus and the decolouring that occurs than other viral viewed decolouring a little less than.

Two kinds of Reichardt dye solutions (80 milligrams/10 milliliters acetonitriles add or do not add 400 milliliters of TWEEN 80 tensio-active agents) have also been prepared.With pipette, extract poliovirus or rhinovirus (all undiluted in substratum) 100 microlitre drops to folding

On the paper handkerchief, and contain viral spot to each and add Reichardt dyestuff drop.For containing tensio-active agent and not containing two kinds of solution of tensio-active agent, color all is quick disappearance.Continue to add dyestuff to color and continue (about 9).Also aforesaid same medium and saline control are tested.Substratum demonstrates certain dye decolored ability really, and the titration behavior that salt solution presents is viewed the same with aforementioned water.

Embodiment 29: the sxemiquantitative of bacterial contamination

Provide the ability of bacterial concentration sxemiquantitative information to confirm to the Reichardt dyestuff.With stationery base material (Neenah Bond ^TM) (available from the NeenahPaper in Georgia State, USA Alpharetta city, Inc.) use first following processing of Reichardt dye solution (80 milligrams/10 milliliters acetonitriles): dip paper in the coating or on paper brushing paint, then paper is dried.Streptococcus aureus (10 with seven concentration known ¹, 10 ², 10 ³, 10 ⁴, 10 ⁵, 10 ⁶With 10 ⁷The CFU/ milliliter) drops in the top of every paper.After about 2 minutes, drop is sucked, being presented at streptococcus aureus concentration is 10 ⁵CFU/mL or when above colour-change obvious.Color distortion is not so obvious during low concentration, particularly for the paper of brushing.

Then carry out blind research.For test objective, be 10 with 100 microlitre concentration ⁶The first drip point of CFU/mL drops in to be dipped on the part that coat paper contains the Reichardt dyestuff.Be 10 with 100 microlitre concentration ⁵The second drip point of CFU/mL drops on the part that the brushing paper contains the Reichardt dyestuff.At last, will contain 10 ⁴The 3rd drip point of the streptococcus aureus of CFU/mL concentration drops in to be dipped on the part that coat paper contains the Reichardt dyestuff.Two experiment participants do not know the concentration of these three drops.After about 2 minutes, drop is sucked.Use control zone, test the separately concentration of each sample of eye estimate of participant for these two.Two people estimate correctly that the concentration of the first sample is 10 ⁶CFU/mL.They guess correctly that also the concentration of the second sample is 10 ⁵CFU/mL.But they are estimated as 10 with the concentration of the 3rd sample improperly ³CFU/mL.It is believed that this mistake at least part of be owing to being lower than 10 ⁵Under the concentration of CFU/mL the color distortion of control zone relatively low due to.But the inventor thinks, can easily select the concentration of chromogen and the homogeneity of coating, to obtain accurate result under this lower concentration.Under any circumstance, since control zone under higher concentration (for example 10 ⁵CFU/mL or more than) more obvious color distortion can be provided, therefore think under clinical more relevant high density, can obtain accurately result.

Embodiment 30: bacterial contamination quantitatively

Provide the ability of bacterial concentration quantitative information to confirm to the Reichardt dyestuff.With stationery base material (Neenah Bond ^TM) (available from NeenahPaper, the Inc. in Georgia State, USA Alpharetta city) and label (available from Avery-Dennison) use first Reichardt dye solution (80 milligrams/10 milliliters acetonitriles) to apply and dry.Use streptococcus aureus, Pseudomonas aeruginosa and colibacillary concentration known aliquots containig (100 microlitre), make the control curve of various bacteriums.More particularly, the bar with coating Reichardt dyestuff is exposed to quantity bacterium aliquots containig decrescence.After applying each aliquots containig, measure the Δ E value of each CFU/mL concentration with hand spectrophotometer and (use L ^*, A ^*And B ^*Value is calculated).The result provides in following table 4 (to paper) and table 5 (to label).

Table 4: the result of stationery base material

log CFU/ml	Δ E (streptococcus aureus)	Δ E (intestinal bacteria)	Δ E (Pseudomonas aeruginosa)
				8	-	9.3642	-
7	11.73368	4.3483	4.9569
				6	3.876455	3.2574	1.3193
5	2.447325	2.3320	1.7151
				4	2.074175	3.0123	2.2358
3	1.866789	3.8228	1.7900

Table 5: the result of label substrate

log CFU/ml	Δ E (streptococcus aureus)	Δ E (intestinal bacteria)	Δ E (Pseudomonas aeruginosa)
				7	18.62321	7.778702	6.9567
6	6.908263	4.866590	4.2419
				5	6.919863	4.643888	4.6519
4	4.791472	5.200596	4.9473
				3	5.413890	5.130312	3.8787

Produce respectively streptococcus aureus, Pseudomonas aeruginosa and colibacillary standard detection curve by above data, shown in Fig. 5-7.As seen, every type of bacterium changes the color of the base material that dyestuff processed in slightly not identical mode, produce unique typical curve among the figure.Afterwards, the drip point with unknown bacterial concentration drops on the stickers the colorific Δ E value of usefulness spectrophotometer measurement.The numerical value that each unknown sample obtains provides in following table 6-7.

Table 6: the result of stationery base material

Table 7: the result of label substrate

Can find out from data, the Δ E value by determining unknown concentration is near which known Δ E value, measurablely goes out this unknown concentration.Although some results are not exclusively accurate, the inventor thinks that the homogeneity of improving coating can further improve accuracy in detection.

Comparing embodiment (the non-embodiment of the invention)

The bacterial origin of chicken as comparing embodiment will be displayed.Half new fresh chicken meat (available from the supermarket), three weeks of at room temperature preservation of transparent film will be covered with on the polystyrene dish.Collect the light yellow juice that compiles in the polystyrene dish with transfer pipet, be used for test.

Comparing embodiment 1:

To display chicken extract puts

On the paper handkerchief.CI Acid Green41 (available from Aldrich Chemical) solution (0.008mol/l) (example of the hydroxyanthraquinone dyestuff) point of following structure 34 dripped to display on the chicken extract.Do not observe colour-change.In contrast, with the 100mgReichardt dye suspension in the 10ml acetonitrile.This suspension point dripped to display on the chicken extract, immediately decolouring.

Comparing embodiment 2:

To display the chicken extract point drips to

On the paper handkerchief.CI AcidGreen 25 solution (0.008mol/l) (example of the anthraquinone dye) point of following structure 35 dripped to display on the chicken extract.Do not observe colour-change.In contrast, with 100mg Reichardt dye suspension in the 10ml acetonitrile.This suspension point dripped to display on the chicken extract, immediately decolouring.

Comparing embodiment 3:

To display the chicken extract point drips to

On the paper handkerchief.The CI Acid Red 37 (available from Aldrich Chemical) (example of amino azoic dyestuff) of the following structure 36 of 50mg is dissolved in the 10ml deionized water.This dye solution point is dripped to displaying on the chicken extract on the paper handkerchief.Do not observe colour-change.In contrast, with 100mg Reichardt dye suspension in the 10ml acetonitrile.This suspension point dripped to display on the chicken extract, immediately decolouring.

Comparing embodiment 4:

To display the chicken extract point drips to

On the paper handkerchief.The CI Acid Yellow 23 (also claiming the food dye lemon yellow) (available from Aldrich Chemical) (example of phenylpyrazole ketone dyes) of the following structure 37 of 50mg is dissolved in the 10ml deionized water.This dye solution point is dripped to displaying on the chicken extract on the paper handkerchief.Do not observe colour-change.In contrast, with the 100mgReichardt dye suspension in the 10ml acetonitrile.This suspension point dripped to display on the chicken extract, immediately decolouring.

Comparing embodiment 5:

To display the chicken extract point drips to

On the paper handkerchief.The aqueous solution point of the CI AcidRed 52 (sulphonyl rhodamine B) (example of xanthene dye) of following structure 38 is dripped to displaying on the chicken extract on the paper handkerchief.Do not observe colour-change.In contrast, with 100mg Reichardt dye suspension in the 10ml acetonitrile.This suspension point dripped to display on the chicken extract, immediately decolouring.

Comparing embodiment 6:

To display the chicken extract point drips to

On the paper handkerchief.The CI Acid Blue 74 (also claiming indigo carmine) (available from Aldrich Chemical) (example of indigoide colors) of the following structure 39 of 30mg is dissolved in the 10ml deionized water.This dye solution point is dripped to displaying on the chicken extract on the paper handkerchief.Do not observe colour-change.In contrast, with 100mg Reichardt dye suspension in the 10ml acetonitrile.This suspension point dripped to display on the chicken extract, immediately decolouring.

Those of skill in the art will recognize that the present invention made various variations and change and to think in those skilled in the art's limit of power.The example of this variation is included in above in this patent of determining, each embodiment by reference integral body is attached to herein, in conjunction with degree be consistent with this specification sheets.The inventor is intended that this variation and change falls within the scope of the present invention.Also will recognize, when reading according to above disclosure, scope of the present invention can not be interpreted as being limited to specific embodiments disclosed herein, the scope of the invention only should be explained according to appending claims.

Claims (14)

1. the method that microorganism exists on a sxemiquantitative or the detection by quantitative surface, described method comprises makes test dyestuff and this Surface Contact, so that detectable colour-change occurs in the test dyestuff, it is characterized in that described test dyestuff comprises zwitterionic N-phenolic acid trimethyl-glycine dyestuff, described N-phenolic acid trimethyl-glycine dyestuff is not 2,6-phenylbenzene-4-(2,4,6-triphenyl pyridine)-phenolate.

2. the method for claim 1, it comprises that also the color with described test dyestuff and contrast dye compares, the color of wherein said contrast dye is corresponding to known microorganisms concentration.

3. the method for claim 2, wherein the color with described test dyestuff and multiple contrast dye compares, and wherein said contrast dye has the color corresponding to different known microorganisms concentration separately.

4. the method for claim 2 further comprises the colour intensity of experiment with measuring dyestuff, one or more contrast dyes or their combination.

5. the concentration of microorganism is directly proportional on the method for claim 4, the colour intensity of wherein said test dyestuff and testing surface.

6. the method for claim 4 further comprises by the colour intensity of one or more contrast dyes is mapped to a plurality of known microorganism concns, produces detection curve.

7. the method for claim 6 further comprises and carries out related with microorganism concn on the institute detection curve colour intensity of described test dyestuff.

8. each method of claim 1 to 7, wherein base material limits the detection zone that contains test materials.

9. each method of claim 1 to 7, wherein said colour-change occurs within being less than approximately 5 minutes.

10. the method for claim 9, wherein said colour-change occur being less than in 1 minute.

11. each method of claim 1 to 7, wherein said test dyestuff is selected from following compound:

Wherein, R be hydrogen ,-C (CH ₃) ₃,-CF ₃Or C ₆F ₁₃

12. base material for microorganism existence in sxemiquantitative or the detection by quantitative sample, described base material limits detection zone, wherein testing dyestuff is included in the detection zone, detectable colour-change can occur in described test dyestuff in the presence of microorganism, it is characterized in that described test dyestuff, and contrast dye or its combination are comprised of zwitterionic N-phenolic acid trimethyl-glycine dyestuff, described N-phenolic acid trimethyl-glycine dyestuff is not 2,6-phenylbenzene-4-(2,4,6-triphenyl pyridine)-phenolate.

13. the base material of claim 12, wherein said test dyestuff is selected from following compound

Wherein, R be hydrogen ,-C (CH ₃) ₃,-CF ₃Or C ₆F ₁₃

14. the base material of claim 12, wherein multiple contrast dye is included in the check plot, and the color of every kind of contrast dye is corresponding to different known microorganisms concentration.

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