Brief description of the results

 

A cross-branch approach has inspired the birth of COSMIC at ENEA since the spring of the year 2000. Ideas and aims of this research have been lined up after “The 2nd Workshop on Chemical Sensors and Biosensors” organised at Enea in 1999, several internal brain storming sessions and enthusiastic seminars from researchers with different expertise (chemists, biochemists, physicists, biologists, molecular biologists, engineers). Acquiring a common language was the priority for combining several intellectual schemes with distinct research tools and for setting up a strategy for smart molecules coupled into chips in the frame of the emerging nanotechnologies, and the newest, ever rising, biosensor and bioelectronic technologies.

This priority was only partly achieved at the begining, on January 2001 when COSMIC started after the agreement of an internal and an external scientific committee of experts, but, during three years, gave all the same successful results which are briefly resumed  in this report. Detailed description of the results can be found in the  full-text paper section.

 

During the reporting period, from January 2001 to December 2003, the activities have been focused on several aspects of the biosensing science, ranging from new functions or activities due to engineered molecules or  genetically modified microorganisms, their oriented and reversible immobilisation on electrodes, optical surfaces and  nano-composite materials,  electrochemical deposition and patterning of protein or oligonucleotide monolayers, real analytical applications.

The title “Coupling Smart Molecules into Chips”  strictly represents the activities in these three years which were focused to handle model biological mediators onto model surfaces made of inorganic materials with a bottom-up approach. Interaction of bioactive proteins with these surfaces, oriented and patterned immobilisation of thin biolayers, diffusion properties of substrates and direct electron transfer were studied with model biomocules, microorganisms and technological materials. The main result of this three year activity is the ability to handle, address and pattern such different model materials and biomaterials for biosensing purposes at the micro and the nano scale  for further development of m-array and m-flow-devices.

      

To get simpler and schematic reading, this report was therefore organised in the following sequential sections which doesn’t reflect the real experimental pathways followed.

 

1. Smart biological materials

 

Several biological materials were obtained from italian and international partners and used as models for their functions or activities:

  • Commercially available purified enzymes (acetyl cholinesterase, alkaline phosphatase, tyrosinase, choline oxidase, glucose oxidase)

  • PSII core complex purified from the thermophilic cyanobacterium Synecococus elongatus (Dr.Jiri Masojidek, Accademy of Science of Czech Republic, Trebon, Czech Republic)

  • Engineered PSII core complex with an his6x-tag from the thermophilic cyanobacterium Synecococus elongatus (dr.Miwa Sugiura, Osaka Prefecture University, Osaka, Japan)

  • scFv of an antibody against the cocumber mosaic virus (CMV)  genetically fused with an alkaline phosphatase activity and an his6x-tag at the C-terminus

  • Adapted and not adapted cells (Pseudomonas putida) (dr.Suna Timur, Bornova University, Izmir, Turkey)

  • Engineered yeasts with a cholinesterase activity from rat expressed onto the cell wall (Prof.Claudio Palleschi, University of Rome “La Sapienza”, Italy)

  

2. Surfaces and Transducers (macro electrodes and m-devices)

 

 Several surfaces and materials were prepared in our laboratories and from our CNR partner:

a.      Commercially available screen printed Au or Pt  surfaces (Krejci Engineering, Czech Republic)

b.      Screen printed carbon graphite composite surfaces doped with:

-commercial metal particles of Rh or Pt on graphite particles  

-nanoparticles of Au supported on graphite nano-particles (Massimo De Francesco, Enea Casaccia, Rome italy) (figure)

-oxidised carbon particles (Roberto Pilloton)

 

c.      Sputtered Au on plastic circles

 

d.      Galvanically deposed gold on copper paths (Roberto Pilloton)

 

e.      m-array of Au on a silicon chip (Dr. Mihaela Ilie University "Politehnica" of Bucharest, Romania, Dr.Vittorio Foglietti CNR-Istituto Nanotecnologie e Fotonica CNR, Rome, Italy)

 

 

Screen printed electrodes (SPE) were extensively used for developing new, low cost, disposable amperometric biosensors. Several lay-outs concerning the arrangement of working, reference and auxiliary electrodes have been proposed. As a rule the working electrode, the reference electrode and the auxiliary, when needed, are printed side by side or concentrically -according to the layout used on the same side of the substrate, being it either a ceramic or a plastic one.

Several original lay-outs were designed and prepared in our laboratories with the  materials listed above to obtain screen printed electrodes with side by side, concentric, front-back or front-back fine toothed comb lay-outs. The global dimensions of the electrodes were decreased by applying the original front to back lay-out: the working and the reference electrodes were, in fact, printed on the opposite sides of a PVC substrate. Quasi-capillary electrodes were obtained, which could eventually be inserted in an hypodermic needle. These electrodes can be used in a two electrode configuration but an external auxiliary electrode can be easily added to the array if needed.

 

 

2. Oriented and reversible immobilisation (electrochemical patterning)

 

His-tagged biomolecules are normally obtained by genetic engineering with the aim of easier purification from bacterial extracts. We used this technique for obtaining oriented immobilisation of biomolecules on electrodes and optical surfaces (glass, quartz, carbon, graphite, silicon, gold). Insertion of the his-tag in a proper part of the biomolecule is responsible of the specific orientation of the active site and other molecule constituents with respect to the electrode surface and the external m-environment.

           

             

 

The ability of certain simple molecules (silanes and thiols) to react with the proper surface (glass, quartz, carbon, graphite, silicon, gold) to give self assembled monolayers (SAM) was used. Starting from SAM of cysteamine on gold or aminopropyltriethoxysilane on hydoxiyated materials, an arm with Ni-Nta chelator was chemically synthesised for further immobilisation of his-tagged proteins (PSII core complex and scFv as model molecules)

 

 

 

Immobilisation based on the natural PSII occurred with the protein A - anti-D1 antibody modified Au electrode.

An electrochemical modification of the 1st step of this synthesis was studied to obtain EDM (Electrochemically deposed monolayers) which greatly improved performances of this immobilisaztion procedure.

Electrochemical formation of CYS layers on Au electrode surface occurred at 0.85V vs RE and resulted in a shorter treatment time (less than 5 min vs 16h of chemical treatment) and in higher Ni+2 surface density. Electrochemical deposition of CYS on Au-graphite composite electrodes occurred at 0.85V vs RE, allowing deposition of CYS only on the metal particles, because of the higher potential (1.2V) needed on graphite electrodes.

With EDM, a redundant Ni-NTA surface density was observed (Ni-NTA:His-tagged-PSII in a ratio of 38000:1) and for this reason, mixed EDM were deposed on the electrodes with different specific roles and functions:

                 I.      immobilisation of the his-tagged proteins (cysteamine, APTS),

                 II.      increased diffusion of molecules to the electrode surface (octadecanthiol),

                 III.      direct electron transfer from redox proteins to the electrode surface (polyaniline).

 

Main results are listed below:

a.      Ni-NTA complex was obtained on several surfaces including carbon, gold and carbon/gold

b.      Immobilised activities of recombinant molecules were verified with photometric, m-spectrofluorimetric and electrochemical methods 

c.      Reversible immobilisation was obtained by using imidazole 200mmol/L. Renewing molecules on the surfaces can be performed during flow analysis in less than 10 min.

d.      Aspecific binding was evaluated with photometric methods for both recombinant molecules

e.      On chip purification of crude bacterial extracts was obtained

f.       Electrochemically addressed immobilisation was obtained on a single Au electrode in a m-array

g.      Mixed SAM of hydrophobic thiols (octadecanthiol) were used for fast diffusion of analytes, inhibitors, products and substrates

h.      Mixed SAM of polyaniline molecular wires for direct electron transfer from redox proteins to the electrode surface were obtained

i.        Specific deposition of different molecules was obtained on Au/carbon composites. Deposition of cysteamine on Au nanoparticles and of polyaniline on carbon was achieved on Au/carbon composites.

These results open the possibility to extend this synthetic procedure to other materials and biomolecules (especially molecular libraries), depending on the specific sensing purpose. All these advantages could achieve the diffusion of reusable, in-situ sensitive, reliable, cheap and easy-to-use sensor systems for environmental analytical application. This technique allows the preparation of GENERIC BIOSENSORS from

  • His-tagged proteins from genetically engineered libraries

  • Antibodies (protein A)

  • Proteins by their specific antibodies (protein A)

 

3. Oxidised carbon particles for proteins immobilisation on screen printed electrodes

 

Over the past few years interest has been increasing in the application of simple, rapid, inexpensive and disposable biosensors in clinical, environmental or industrial analysis. The most common disposable biosensors are those produced by thick-film technology. A thick-film biosensor configuration is normally considered to be one which comprises layers of special inks (or pastes) deposited sequentially onto an insulating support or substrate. Screen printing seems to be one of the most promising technologies allowing biosensor to be placed large-scale on the market in the near future because of advantages such as miniaturisation, versatility at low cost and also particularly the possibility of mass productions. The use of thick-film technology for the production of sensor systems is an emerging field. The most critical point in manufacturing thick film biosensors is the sensing or active membrane and its adhesion to the transducer layer.   Activation and derivatization of carbon powders to be used in screen printed electrode preparation can be performed with an oxidizing treatment of  carbon powder with aqueous hydrogen peroxide which introduces mainly carboxylic and phenolic groups on the carbon surface after that as well as derivatization, it is easy to use this groups in covalent immobilization of  biomolecules.  Carbon particles oxidised for 48 h in a hydrogen peroxide bath were then used for printing electrodes with functional groups on the surface available for further one step immobilisation of enzymes.

The oxidizing treatment of carbon particles modified the surface chemistry of the electrode, by creating surface-oxygen complexes that make it more acidic. Scanning electron microscopy showed good evidence that the porous structure of the carbonaceous support was not affected  by the treatment. Furthermore, the pH value of the carbon slurry was found to be changed from 6.5 to 4.4 after oxidizing step because of the presence of acidic groups on the surface. On the other hand, as well as the covalent immobilization by using the introduced acidic groups, negatively charged functional groups on the electrode surfaces could provide useful matrices for covalent or ionic binding of proteins which could strongly interact in aqueous solution. In this study, diferent oxidised carbon matrices (graphite-rhodium, carbon, carbon-dextran and lysine coupled carbon) were prepared to be printed as a working electrode.  Afterwards, covalent immobilization of glucose oxidase was performed. Glucose oxidase was used as a model enzyme to test the usage of the different covalent immobilization methods on the treated carbon surfaces.

 

4. possible Application of Screen Printed Electrodes to real analytical problems

 

Recently, biosensor research was not only oriented to improve analytical performances but also focused on mass production, lower manufacturing costs and reproducibility. Screen-printing was chosen as one of the most promising technology, allowing biosensors to be largely on the market especially with disposable devices. Additionally, the development of new biosensors based on genetically engineered molecules may help to fulfill such requirements providing new reagents at high yields and low cost. In this perspective, the availability of repertoires of recombinant antibodies displayed on phage may allow the quick isolation of new binding molecules with high specificity to virtually any analyte. Nowadays, recombinant molecules or microrganisms by using TFT allow to combine the extremely high specific activity of the former with the advantages from the short time of analysis, the low production costs, and the good reproducibility of disposable SPEs.

Pesticides are largely diffused in agriculture because of their high efficiency and relatively rapid degradation in environment, but their distribution and recycling processes result in water and soil pollution, with dangerous and acute effects on the living organisms, i.e. altering the food chain, or inhibiting the active site of fundamental enzymes, as well as cholinesterases involved in muscle physiology and in nervous system. On the other hand triazine herbicides, which continue to be used every year in large amounts, inhibit the photosynthetic activity of plants and show dangerous carcinogenic effects on mammalians. Some of these compounds, like dinoseb and atrazine, were banned in most countries for their certified toxicity, whilst the European Drinking Water Act (1980) does not allow their concentration in drinking water to exceed individually the limit of 0.1 mg/L. Analytical monitoring of such a low level with both high sensitivity and selectivity remains a topical issue, especially when in presence of interfering compounds. HPLC, GC-MS, ELISA methods were showed to be expensive in instrumentation and/or relatively difficult, time consuming in the sample treatment, although highly sensitive. Furthermore, most of these methods do not provide any information about toxicity and effects on living organisms. These concerns have stimulated research towards development of biosensing technology as a new tool for detecting herbicide and insecticide toxicity in a simple and cost effective way by simply measuring the residual activity of several enzymes after the exposure to several classes of pollutants. Several AChE and OPH based biosensors were already developed to detect paraoxon, methyl-paration and diazinon.

Inhibition based biosensors for herbicides, organophosphorous and carbamic pesticides, phenols and free radicals were studied by coupling screen printed electrodes with several enzyme or whole cells systems as well as acetylcholinesterase, alkaline phosphatase, natural and engineered PSII, engineered yeasts and adapted cells.

 

a. AchE based biosensors for pesticides detection: Residual activity of free or immobilised AcChE located on the biosensor tip was normally detected in the real sample where incubation with OP was performed. Serious problems took place due to:

i) adsorbed AcChE,

ii) electrochemical interferences from the sample and

iii) very low lifetime of the biosensor.

AcChE immobilised on a separate membrane placed in solution, provided a different protocol of analysis with separation between incubation step and final measurement in a clean standard solution allowing the biosensor to be used without any protecting membrane. A very high reproducibility for paraoxon in drinkable water, lower analysis time (80s/sample with parallel operation on 24 samples) and the possibility to use the same ChOx biosensor for several hundreds of analyses, were achieved.

A protocol for anticholinesterase activity on grapes was then developed which well applies to the concentration range of pesticides normally on fruit and to the limit fixed by the European regulation. This procedure was extended to a biosensing system based on a recombinant yeast with a genetically expressed AcChE activity from rat. All these advantages could achieve the diffusion of reusable, in-situ, sensitive, reliable, cheap and easy-to-use sensors for environmental analytical application.

 

b. AcP based biosensors for pesticides detection: Performances of AcP based screen printed electrodes prove a close correlation with previously obtained analogous bienzymatic biosensor, with the advantage of the requirement of only a single enzyme.. Another advantage of the AcP based sensor with respect to the cholinesterase based sensor is that the inhibition of AcP by organophosphorous pesticides is almost completely reversible, so that no reactivating treatment is required. The reversibility of the inhibition of AcP, in the case of the immobilized enzyme biosensor, could be also responsible for the relatively long shelf life of the sensor, leading to a drastic reduction of the overall costs of operation.

 

c. PSII based biosensors for herbicide detection: The herbicides inhibiting PSII still represent a basic tool of weed control. Their overuse brings serious environmental and health risks. Since chromatographic methods and immunoassays are not suitable for prescreening assay, we have recently designed a biosensor based on isolated PSII from thermophilic cyanobacteria Synechococcus elongatus. A stable and sensitive semi-automated biosensor for detection of triazine-type herbicides was developed, using isolated PSII particles immobilized on a Clark electrode.  This biosensor exhibited a good stability as well as high sensitivity, achieving a LOD=5x10-10 M for diuron. However the use of Clark electrodes prevents mass production, thus printing technology, useful in batch production of low price electrochemical devices, was initially adopted with the purified wild-type PSII. Printed electrodes and recombinant PSII were finally coupled with several advantages concerning sensitivity, stability, life time and response time.

 

d. Whole cell biosensors for phenols detection: Biotechnological processes have been employed in several industrial productions, in biomedical applications and in environmental remediation. Interest was focused on biomediators as purified enzymes or metabolic pathways of cells, tissues etc. with specific biochemical properties for analytical and either production or degradation applications. In this frame Pseudomonas putida based biosensors for determining the phenolic compounds were dveloped. The intensive increase in the release of a diverse range of hazardous compounds into the environment has made their detection of paramount importance. In particular, this is necessary for successful clean-up of pollution from the environment and timely elimination of the consequences or a reduction of the scale of hazardous release. Phenolic compounds are one of the major pollutants of industrial waste waters and, at the same time, the compounds that have high toxicity to the human organism when present above certain concentration limits, which has required for rapid, easy to operate, low-cost toxicity screening procedures. The trend towards simplification and automation of the analysis methods used in modern chemical and biochemical laboratories or in quality control of some industrial biosynthesis processes has led to setting up some electrometric procedures for determining phenol, based on biosensors. The use of microbial biosensors to determine the concentrations of substances is based on the presence of specific enzyme systems in microorganisms which transform certain chemical compounds. The transformation processes can be accompanied by the appearance of electrochemically active products or utilization of reaction co-substrates, which enable the use of standard electrochemical techniques -amperometry or potentiometry - for their registration. As judged by their sensitivity, time of response and stability of signals, microbial sensors are similar to enzyme based sensors but are less selective. This may be due to the complexity of the elements of the enzyme apparatus of cells. Insignificant amount of biomass as well as high stability make the use of microbial sensors preferable in some cases compared to enzyme sensors. This is especially true in the detection of a pool of toxic compounds showing similar composition, or the assessment of comprehensive indices of the condition of the environment as, for instance, biological oxygen demand (BOD). Amperometric biosensors using bacterial cells were developed for the detection of phenolic compounds. Pseudomonas putida DSM 50026 was used as a biological component and the measurement was based on the respiratory activity of the cells. For this purpose, the cells were grown in the presence of phenol as the sole source of organic carbon. As well as phenol adapted cells, the bacterium which used the glucose as the major carbon source, was also used to obtain another type of biosensor for the comparison of the responses and specificities towards to different xenobiotics.

 

Bacterial cells with and without adaptation steps in a medium with phenol as the only carbon source enabled us to get biosensor systems with different substrate specificities, thus it could be possible to analyze different phenolic substances individually in the mixed samples  by using these sensors. In previous studies, different types of microorganisms were used in biosensor systems. In comparison to the others, our biosensors have higher sensitivity. Furthermore, immobilization of microorganisms instead of pure enzymes on the thick film electrodes provided economic and practical disposable biosensors. The immobilization method was also useful for the protection of microbial activity. All our findings showed that the obtained biosensors could be used as a simple, rapid and direct methods of determining phenol in various media such as waste water samples with different compositions.

 

e. Disposable screen printed potentiometric sensors for detection of free radicals: Recently, low cost and rapid analytical determinations of free radicals are playing a central role in environment and food monitoring. Screen printed potentiometric sensors based on an ionophore dispersed in a PVC membrane were previously verified and optimised by using valinomycin as model molecule for potassium ion activity measurement. Valinomycin was used for finding the best deposition conditions of the PVC membrane directly on the printed graphite electrodes. Valinomycin containing membranes of different thickness were obtained with “dip & dry” or “tape casting” or “screen printing” techniques by using different dilution of the PVC matrix in tetrahydrofurane (THF). Screen printing of the PVC membranes gave the worse results and was abandoned. With dip & dry and casting procedures, sensitivity of the valinomicyn based sensors for K+ was found to be strictly related to the thickness of the membrane and the used deposition technique. A very good reproducibility was also observed by comparing the slope values of the calibration curves obtained from several electrodes.

The development of disposable, screen printed potentiometric sensors for free radicals, was based on a nitrone species dispersed in the PVC membrane. Nitrone is able to give spin trapping reactions with free radicals and, for this reason, a membrane potential variation can be observed. PVC/nitrone coated electrodes (dip & dry) were tested in EDTA 0.02 mol/L, pH=5.16, FeCl2 0.02 mol/L and known amounts of H2O2 0.1 mol/L were added for hydroxide radical production. A greatly over-Nernstian slope in the calibration curves was observed with high reproducibility. This effect could be due to the propagation of the radical based reaction which may result in an amplification of the signal but, at the moment, additional, interfering reactions cannot be excluded. PVC/nitrone coated electrodes (dip & dry) were also used with the xanthine/xanthine oxidase method for superoxide radical production. The slope of the calibration curve was again over-Nernstian but lower than in the previous experiments and nearer to the expected value.