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BIOMOLECULAR ADSORPTION AND THE LIFEDETECTOR

J. Panitz

To cite this version:J. Panitz. BIOMOLECULAR ADSORPTION AND THE LIFE DETECTOR. Journal de PhysiqueColloques, 1984, 45 (C9), pp.C9-285-C6-291. �10.1051/jphyscol:1984948�. �jpa-00224429�

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JOURNAL DE PHYSIQUE

Colloque C9, supplément au n°12, Tome *5, décembre 1984 page C9-285

BIOMOLECULAR ADSORPTION AND THE LIFE DETECTOR

J.A. Panitz

Sandia National Laboratories, Albuquerque, NM 87185, U.S.A.

Résume: Ce mémoire traite du dépôt de molécules biologi-ques sur la surface de pointes métalliques a émission de champ. Une nouvelle procédure de dépôt et une nouvelle méthode de coloration permettent de déposer des protéines avec précision sur 1' extrémité' d'une pointe, pour les visualiser ensuite dans le microscope électronique à transmission. Ces techniques permettent la préparation et la caractérisation de pointes à émission de champ immunologiquement actives. En combinant la spécificité chimique de ces pointes aux possibilités d'amplification moléculaire propres au processus d'émission par effet de champ, on peut alors envisager la création d'un détecteur chimique d'une conception entièrement nouvelle.

Abstract: The placement of biological molecules on the surface of metal field-emitter tips is discussed. A new deposition protocol and staining procedure allows precise coverages of proteins to be placed on the apex of a tip and then visualized in the transmission electron microscope. These techniques provide a means for preparing and then characterizing immuno-logically active field-emitter tips. By combining the chemical specificity of these tips with the molecular amplification capabilities of the field-electron emission process, a novel chemical sensor concept can be envisioned.

Introduction: For several years we have discussed methods to place biological macromolecules on the apex of a metal field-emitter tip [1-4]. These studies were motivated by the development of a field-ion Tomographic (FIT) microscope that used point-projection imaging to reveal the three-dimensional appearance of unstained biological molecules[5]. The images that we obtained demonstrated the feasibil-ity of the technique [6-7], but image reproducibility was poor, presumeably because the nature of the deposition process encouraged the binding of molecular aggregates of random size and shape — rather than single molecules — to the region of the tip apex acces-sible to imaging. We demonstrated that ferritin could be visualized on the surface of metal field-emitter tips by imaging the tips in profile in the TEM[4]. The core of an individual ferritin molecule could be seen because it contains a high concentration of iron, an electron opague element. Clusters of ferritin were always observed which seemed to confirm our conclusion that the reproducibility of a FIT image was related to the size, the shape and the distribution of ferritin clusters on the tip apex.

Attempts to image DNA by field-ion tomography were moderately suc-cessful^], even though DNA could not be independently visualized

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1984948

http://www.edpsciences.orghttp://dx.doi.org/10.1051/jphyscol:1984948

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on a t i p by TEM imaging because DNA i s not e l e c t r o n opaque. Without TEM imaging, t r i a l and e r r o r , and an a p r i o r i knowledge (and b e l i e f ) i n t h e appearance of a FIT image was requi red t o develop a succes s fu l depos i t i on s t r a t e g y .

Rotary s t a in ing : The problem of depos i t i ng a b io log ica l molecule on a t i p apex is complicated by t h e i n t e r a c t i o n of many parameters . Bio logica l molecules must be depos i ted from aqueous so lu t ion . The type of b u f f e r t h a t is used, its pH and t h e concent ra t ion of o t h e r c o n s t i t u e n t s (such a s s a l t ) , t h e a d v i s a b i l i t y of " f i x ing" t h e mole- cu l e s i n solut ion[81, and even t h e temperature of t h e depos i t i on s o l u t i o n can have an e f f e c t on t h e coverage and t h e morphology of t h e molecules t h a t a r e placed on t h e t i p sur face . In order t o evalu- a t e t h e importance of t h e s e parameters, it was e s s e n t i a l t o develop a gene r i c procedure f o r v i s u a l i z i n g t h e coverage of any molecule on t h e t i p su r f ace by d i r e c t imaging i n t h e TEM. This meant t h a t t h e image c o n t r a s t of an adsorbed molecule had t o be increased . The procedure t h a t we developed t o accomplish t h i s t a s k is shown schema- t i c a l l y i n Figure 1[9].

ROTARY

HOLD

TABL

Fig. 1. A procedure f o r r o t a r y s t a i n i n g f i e ld -emi t t e r t i p s .

Af t e r p lac ing molecules on t h e t i p sur face , t h e t i p i s r o t a t e d i n vacuum and covered with a t h i n l a y e r of evaporated metal. The 1 nm - 2 nm t h i c k l a y e r enhances t h e v i s i b i l i t y of i nd iv idua l molecules i n a TEM image by enc los ing them wi th in an e l e c t r o n opaque s h e l l t h a t f a i t h f u l l y preserves t h e i r shape. Our procedure is a c t u a l l y an ex- t ens ion of t h e well known r o t a r y s t a i n i n g process used by molecular b i o l o g i s t s t o enhance t h e c o n t r a s t of p r o t e i n s and nuc l e i c a c i d s placed on t h i n carbon f i lms and imaged i n t h e TEMClO].

Figure 2 shows t h e r e s u l t o f r o t a r y s t a i n i n g a p a r t i a l monolayer o f f e r r i t i n molecules with tungsten. The p r o t e i n s h e l l of i nd iv idua l f e r r i t i n molecules is c l e a r l y seen, de l inea t ed by a t h i n l a y e r o f evaporated tungsten. Indiv idua l molecules t h a t a r e behind one another i n t h e imaging d i r e c t i o n a r e a l s o v i sua l i zed . The 200 kV e l e c t r o n beam used fo r imaging e a s i l y p e n e t r a t e s each molecule, reproducing d e t a i l from each spec i e s encountered on its way t o t h e photographic emulsion. Electron beam damage is minimized by t h e s h o r t dwell t ime of t h e e l e c t r o n s i n an adsorbed spec ies .

F i g . 2. F e r r i t i n molecules r o t a r y s t a i n e d wi th t u n g s t e n .

Rota ry s t a i n i n g h a s allowed us t o deve lop a d e p o s i t i o n p r o t o c o l t h a t is c a p a b l e o f p l a c i n g a r e p r o d u c i b l e coverage o f molecules on t h e s u r f a c e o f a f i e l d - e m i t t e r t i p c l l ] . The e s s e n t i a l s t e p s i n t h e p r o t o c o l are as fo l lows :

1. Mo1,ecules are d e p o s i t e d o n t o a t i p f o r two minutes from a 1 0 p l d r o p l e t of b u f f e r t h a t c o n t a i n s t h e molecule a t t h e d e s i r e d c o n c e n t r a t i o n . A t y p i c a l b u f f e r f o r p r o t e i n depos i - t i o n c o n s i s t s o f 50mM HEPES, 150mM NaCl (pH 7 .5 ) .

2. The t i p is r i n s e d f o r two minu tes i n l O m l o f b u f f e r . At t h i s p o i n t , d e p o s i t i o n s t o p s because t h e c o n c e n t r a t i o n o f molecu les i n s o l u t i o n h a s been reduced by t h e d r o p l e t / b u f f e r volume r a t i o (10/10000 = .001) .

3 . Molecules t h a t a r e adsorbed on t h e t i p s u r f a c e a r e f i x e d f o r f i v e minu tes i n a 20pl d r o p l e t o f a 0.6% s o l u t i ~ n o f g l u t e r - a ldehyde i n b u f f e r .

4. The t i p is r i n s e d f o r one minute i n l O m l o f f r e s h , doub le ( g l a s s ) d i s t i l l e d wa te r .

5. The t i p is r i n s e d f o r one minute i n l O m l of a 20% mix ture o f doub le ( g l a s s ) d i s t i l l e d e t h a n o l i n doub le d i s t i l l e d wa te r .

6. The t i p is r i n s e d f o r one minute i n l O m l o f a 50% mix ture o f doub le d i s t i l l e d e t h a n o l i n doub le d i s t i l l e d wa te r .

7. The t i p is r i n s e d f o r one minute i n l O m l of a 70% mix ture o f doub le d i s t i l l e d e t h a n o l i n doub le d i s t i l l e d wa te r .

8. The t i p is r i n s e d f o r one minute i n l O m l o f 100% double d i s t i l l e d e t h a n o l , removed i n t o a i r , and d r i e d .

I t is e s s e n t i a l t o i n s u r e t h a t t h e t i p remains wet u n t i l t h e r i n s e i n 100% e t h a n o l h a s been completed. A s p e c i a l f i x t u r e h a s been des igned f o r t h i s purpose[ l l ] . With our p r o t o c o l , the a b i l i t y t o a d s o r b i s o - l a t e d prote- in molecules on t h e t i p apex depends upon t h e l a c k of molecu la r a g g r e g a t i o n i n s o l u t i o n p r i o r t o d e p o s i t i o n . Aggregat ion on t h e t i p s u r f a c e is minimized by t h e r i n s e schedu le o u t l i n e d above.

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The sequence of ethanol-water mixtures g radua l ly r e2 l aces t h e i n i t i a l aqueous environment of t h e molecule wi th pure e thanol . This reduces t h e su r f ace t ens ion fo rces t h a t a c t on t h e adsorbed molecules a s t h e t i p is removed i n t o a i r and dr ied . I f a t i p i s removed i n t o a i r from pure water , t h e number and t h e d i s t r i b u t i o n of 'molecules depos i ted on its apex w i l l be s eve re ly perturbed, and l a t e r a l aggrega t ion w i l l in - c r e a s e [ l l ] . Immunologic de t ec t ion : Severa l years ago, we used t h e TEM t o v i s u a l i z e t h e morphology of t h i c k p r o t e i n l a y e r s placed on a t i p by t h e immune reactionC31. Unstained p r o t e i n l a y e r s could be seen covering t h e t i p su r f ace because the d e n s i t y of t h e depos i t and its th i ckness insured t h a t s u f f i c i e n t e l e c t r o n s c a t t e r i n g would occur, and adequate image c o n t r a s t would be obtained. Recently, w e decided t o use our new depos i t i on and shadowing techniques t o i n v e s t i g a t e , i n more de- t a i l , p r o t e i n mu l t i l aye r s formed on t h e t i p su r f ace by t h e immune r eac t ion . In p a r t i c u l a r , we wanted t o see i f a chemical ly s p e c i f i c su r f ace could be prepared by adsorbing a s e l ec t ed p r o t e i n an t igen on t h e t i p apex, and then s e l e c t i v e l y captur ing i t s s p e c i f i c p r o t e i n ant ibody from so lu t ion by t h e immune r eac t ion . Figure 3 shows a t i p on which a monolayer of an t igen ( f e r r i t i n ) ha s been p laced . F igure 4 shows t h e mu l t i l aye r formed when a f e r r i t i n coated t i p is exposed t o a s o l u t i o n conta in ing a low concent ra t ion of a n t i - f e r r i t i n r a b b i t an t ibod ie s of t h e IgG type.

Fig. 3. A s a t u r a t i o n coverage of f e r r i t i n .

Fig. 4. A n t i - f e r r i t i n r a b b i t IgG bound t o f e r r i t i n .

The difference in layer morphology between Figure 3 and Figure 4 is caused by the formation of immune complexes after exposing the antigen covered tip to antibody. A series of such experiments has demon- strated that it is possible to prepare an immunologically active tip that will recognize and irreversibly bind antibody from a solution containing other contaminant proteins.

The exact mechanism that is responsible for the highly specific recog- nition phenomena between antigen and antibody molecules is largely unknown. Chemical and steric interactions over angstrom dimensions, coupled with morphological changes in one or both species, are pro- bably responsible for the tight binding which is generally observed [12]. The rate constant for the dissociation of an antigen-antibody complex in solution is typically six orders of magnitude smaller than the rate constant that is associated with its formation[12].

The LIFE Detector: Our experience in preparing immunologically ac- tive field-emitter tips has suggested a novel chemical sensor concept. It combines the chemical specificity of an immunologically active field-emitter tip with the single molecule detection capability of the field-electron emission process. Field-electron emission in vacuum can be used as a nonspecific detector of biomolecular adsorption from aqueous solution[ 131. It is also known that field-electron emission can be observed in very pure, cryogenic liquids[14]. By operating a field-emission diode in a biologically compatable fluid at room tem- perature, with an immunologically active field-emitter tip, a chemical sensor might be created with a single molecule detection capability. The sensor would be very small, lightweight, and require almost no power for operation. It could be tailored to detect almost any chemi- cal species in solution by binding its complementory antigen (or anti- body) to the surface of a field-emitter tip. The resulting LIquid Field-Emission (or, LIFE) detector would be a biologically active diode. The concept is shown schematically in Figure 5 .

Fig. 5. The LIFE (LIquid Field-Emission) detector concept.

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F i g u r e 5 d e p i c t s a n a n t i g e n covered t i p t h a t has i r r e v e r s i b l y bound a n t i b o d y from s o l u t i o n . I n a c t i v e r e g i o n s o f t h e t i p s u r f a c e are p a s s i v a t e d a g a i n s t n o n s p e c i f i c a d s o r p t i o n . When t h e t i p is b i a s e d t o a n e g a t i v e p o t e n t i a l , a c u r r e n t i s drawn t h a t w i l l r e f l e c t t h e coverage of molecules bound t o t h e t i p apex. We are e v a l u a t i n g s e v e r a l l i q u i d s i n which s t a b l e e l e c t r o n c u r r e n t s can b e drawn a t room tempera tu re , and i n which immunological a c t i v i t y can be main- t a i n e d . Promising c a n d i d a t e s a r e mix tu res of d imethyl s u l f o x i d e (DMSO) and wate r . S t a b l e e l e c t r o n c u r r e n t s have been measured a t room t e m p e r a t u r e from t i p s coa ted w i t h bov ine serum albumin (BSA), and w i t h f e r r i t i n con juga ted t o g o a t a n t i - r a b b i t I g G . A t i p imaged i n t h e TEM before , and a f t e r a t y p i c a l c u r r e n t measurement is shown i n f i g u r e 6A and F i g u r e 68, r e s p e c t i v e l y . The f i x t u r e t h a t we used t o make t h e c u r r e n t measurements i s shown i n F i g u r e 6C. An e l e c t r o - meter connected t o t h e anode measured t h e c u r r e n t i n j e c t e d i n t o t h e

F i g u r e 6. C u r r e n t i n j e c t i o n i n t o a l i q u i d .

liquid from the molecule-coated tip. Figure 6D shows a typical cur- rent profile measured by applying a bias of -273 Vdc to an antigen (BSA) covered tip in a 10% mixture of DMSO in water. Figure 6A and Figure 6B demonstrate that the morphology of the tip and the appear- ance of the BSA layer on its surface did not change as a result of drawing current from the tip for several minutes. After drawing cur- rent for about ten minutes, the current profile became erratic; after about twenty minutes the average current increased catastropi- cally, and the tip was quickly destroyed by electrochemical erosion. The same effect has been observed on several tips, and has been associated with a gradual decomposition of the BSA layer that ini- tially coats its surface. The random current spikes seen in Figure 6C may reflect a series of minor electrochemical breakdown events prior to complete destruction of the protein layer. On the other hand, they may reflect the adsorption and desorption of molecular contaminants from the liquid at the BSA-liquid interface. Future studies will attempt to clarify the mechanism that produces the measured current, and the sensitivity of the measured current to con- taminant binding at the surface of the tip. Current profiles will also be measured as a function of antibody concentration in solution.

Conclusions: A new deposition protocol and a new rotary staining process have resulted in the preparation and the characterization of immunologically active field-emitter tips. A novel chemical sensor concept has been introduced in which the chemically specificity of the immune reaction is combined with the single molecule detection capability of the field-electron emission process. Stable electron currents have been drawn from tips covered with a protein layer and immersed in a biologically compatable liquid at room temperature. Experiments are planned to test the sensitivity of the detector to the adsorption of antibody from solution, and to optimize the liquid environment in which the detector operates.

Acknowledgement: The author would like to thank the Defense Advanced Research Projects Agency (DARPA) who supported this work under ARPA grant # 4597.

References:

c11 Panitz, J.A. and Giaever, I. Ultramiscoscopy. _4 (1979) 361. C21 Panitz, J.A. and Giaever, I. Ultramicroscopy. 5 (1980) 284. C31 Panitz, J.A. and Giaever, I. Surface Science. 97 (1980) 25. C41 Panitz, J.A. and Giaever, I. Ultramicroscopy. 6 (1981) 3. [ 51 Panitz, J.A., J. Micros. 125 (1982) 1. C61 Panitz, J.A., Ultramicroscopy. 7 (1982) 241 [ 71 Panitz, J.A., Ultramicroscopy. 11 (1983) 161. c81 Bullock, G.R., J. Micros. 133 (-84) 1. [91 Panitz, J.A., J. Micros. (inpress). [lo] Griffith, J.D., In: Methods of Cell Biology (ed. D. M. Prescott)

7 p. 129. Academic Press, New York, 1973. [11] Panitz, J.A., Andrews, C.L. and Bear, D.B., J. Electron Micros.

Technique (in press). El21 Ward, F.A., A Primer of Immunology u utter worth, London, 1970). [I31 Panitz, J.A., J. Appl. Phys. (in press). C141 Halpern, B. and Gomer, R., J. Chem. Phys. 2 (1969) 1031.