Antonia Mallardi

Electrolyte‐Gated Organic Field‐Effect Transistor Sensors Based on Supported Biotinylated Phospholipid Bilayer

Anchored, biotinylated phospholipids forming the capturing layers in an electrolyte-gated organic field-effect transistor (EGOFET) allow label-free electronic specific detection at a concentration level of 10 nM in a high ionic strength solution. The sensing mechanism is based on a clear capacitive effect across the PL layers involving the charges of the target molecules.

Carbon based materials for electronic bio-sensing

Bio-sensing represents one of the most attractive applications of carbon material based electronic devices; nevertheless, the complete integration of bioactive transducing elements still represents a major challenge, particularly in terms of preserving biological function and specificity while maintaining the sensors electronic performance. This review highlights recent advances in the realization of field-effect transistor (FET) based sensors that comprise a bio-receptor within the FET channel. A birds-eye view will be provided of the most promising classes of active layers as well as different device architectures and methods of fabrication. Finally, strategies for interfacing bio-components with organic or carbon nano-structured electronic active layers are reported.

Interfacial electronic effects in functional biolayers integrated into organic field-effect transistors

Biosystems integration into an organic field-effect transistor (OFET) structure is achieved by spin coating phospholipid or protein layers between the gate dielectric and the organic semiconductor. An architecture directly interfacing supported biological layers to the OFET channel is proposed and, strikingly, both the electronic properties and the biointerlayer functionality are fully retained. The platform bench tests involved OFETs integrating phospholipids and bacteriorhodopsin exposed to 1–5% anesthetic doses that reveal drug-induced changes in the lipid membrane. This result challenges the current anesthetic action model relying on the so far provided evidence that doses much higher than clinically relevant ones (2.4%) do not alter lipid bilayers’ structure significantly. Furthermore, a streptavidin embedding OFET shows label-free biotin electronic detection at 10 parts-per-trillion concentration level, reaching state-of-the-art fluorescent assay performances. These examples show how the proposed bioelectronic platform, besides resulting in extremely performing biosensors, can open insights into biologically relevant phenomena involving membrane weak interfacial modifications.

Electron Transfer Kinetics in Photosynthetic Reaction Centers Embedded in Trehalose Glasses: Trapping of Conformational Substates at Room Temperature

We report on room temperature electron transfer in the reaction center (RC) complex purified from Rhodobacter sphaeroides. The protein was embedded in trehalose-water systems of different trehalose/water ratios. This enabled us to get new insights on the relationship between RC conformational dynamics and long-range electron transfer. In particular, we measured the kinetics of electron transfer from the primary reduced quinone acceptor (Q(A)(-)) to the primary photo oxidized donor (P(+)), by time-resolved absorption spectroscopy, as a function of the matrix composition. The composition was evaluated either by weighing (liquid samples) or by near infrared spectroscopy (highly viscous or solid glasses). Deconvolution of the observed, nonexponential kinetics required a continuous spectrum of rate constants. The average rate constant ( = 8.7 s(-1) in a 28% (w/w) trehalose solution) increases smoothly by increasing the trehalose/water ratio. In solid glasses, at trehalose/water ratios > or = 97%, an abrupt increase is observed ( = 26.6 s(-1) in the driest solid sample). A dramatic broadening of the rate distribution function parallels the above sudden increase. Both effects fully revert upon rehydration of the glass. We compared the kinetics observed at room temperature in extensively dried water-trehalose matrices with the ones measured in glycerol-water mixtures at cryogenic temperatures and conclude that, in solid trehalose-water glasses, the thermal fluctuations among conformational substates are inhibited. This was inferred from the large broadening of the rate constant distribution for electron transfer obtained in solid glasses, which was due to the free energy distribution barriers having become quasi static. Accordingly, the RC relaxation from dark-adapted to light-adapted conformation, which follows primary charge separation at room temperature, is progressively hindered over the time scale of P(+)Q(A)(-) charge recombination, upon decreasing the water content. In solid trehalose-water glasses the electron transfer process resulted much more affected than in RC dried in the absence of sugar. This indicated a larger hindering of the internal dynamics in trehalose-coated RC, notwithstanding the larger amount of residual water present in comparison with samples dried in the absence of sugar.

Detection beyond Debye's length with an electrolyte-gated organic field-effect transistor.

Electrolyte-gated organic field-effect transistors are successfully used as biosensors to detect binding events occurring at distances from the transistor electronic channel that are much larger than the Debye length in highly concentrated solutions. The sensing mechanism is mainly capacitive and is due to the formation of Donnans equilibria within the protein layer, leading to an extra capacitance (CDON) in series to the gating system.

Development of a novel enzyme/semiconductor nanoparticles system for biosensor application

Abstract Nanosized semiconductor crystals can increase efficiency of photochemical reactions and can be effectively coupled to biomolecular units, such as enzyme, to generate novel photoelectrochemical systems. In this work, nanocrystalline CdS has been synthesized by using a microemulsive system and immobilised by self-assembling on a gold electrode in order to prepare, combined with formaldehyde dehydrogenase (FDH) enzyme, a biological-inorganic hybrid able to perform catalytic oxidation of formaldehyde. The preliminary results indicate that quantum-sized CdS layer on gold, in close contact with the enzyme, is an effective photoactive material to replace the NAD+/NADH role as charge transfer in the enzymatic reaction.

Part per trillion label-free electronic bioanalytical detection

A Functional Bio-Interlayer Organic Field-Effect Transistor (FBI-OFET) sensor, embedding a streptavidin protein capturing layer, capable of performing label-free selective electronic detection of biotin at 3 part per trillion (mass fraction) or 15 pM, is proposed here. The response shows a logarithmic dependence spanning over 5 orders of magnitude of analyte concentration. The optimization of the FBI analytical performances is achieved by depositing the capturing layer through a controllable Layer-by-Layer (LbL) assembly, while an easy processable spin-coating deposition is proposed for potential low-cost production of equally highly performing sensors. Furthermore, a Langmuirian adsorption based model allows rationalizing the analyte binding process to the capturing layer. The FBI-OFET device is shown to operate also with an antibody interlayer as well as with an ad hoc designed microfluidic system. These occurrences, along with the proven extremely high sensitivity and selectivity, open to FBI-OFETs consideration as disposable electronic strip-tests for assays in biological fluids requiring very low detection limits.

Residual Water Modulates QA−-to-QB Electron Transfer in Bacterial Reaction Centers Embedded in Trehalose Amorphous Matrices

The role of protein dynamics in the electron transfer from the reduced primary quinone, Q(A)(-), to the secondary quinone, Q(B), was studied at room temperature in isolated reaction centers (RC) from the photosynthetic bacterium Rhodobacter sphaeroides by incorporating the protein in trehalose water systems of different trehalose/water ratios. The effects of dehydration on the reaction kinetics were examined by analyzing charge recombination after different regimes of RC photoexcitation (single laser pulse, double flash, and continuous light) as well as by monitoring flash-induced electrochromic effects in the near infrared spectral region. Independent approaches show that dehydration of RC-containing matrices causes reversible, inhomogeneous inhibition of Q(A)(-)-to-Q(B) electron transfer, involving two subpopulations of RCs. In one of these populations (i.e., active), the electron transfer to Q(B) is slowed but still successfully competing with P(+)Q(A)(-) recombination, even in the driest samples; in the other (i.e., inactive), electron transfer to Q(B) after a laser pulse is hindered, inasmuch as only recombination of the P(+)Q(A)(-) state is observed. Small residual water variations ( approximately 7 wt %) modulate fully the relative fraction of the two populations, with the active one decreasing to zero in the driest samples. Analysis of charge recombination after continuous illumination indicates that, in the inactive subpopulation, the conformational changes that rate-limit electron transfer can be slowed by >4 orders of magnitude. The reported effects are consistent with conformational gating of the reaction and demonstrate that the conformational dynamics controlling electron transfer to Q(B) is strongly enslaved to the structure and dynamics of the surrounding medium. Comparing the effects of dehydration on P(+)Q(A)(-)-->PQ(A) recombination and Q(A)(-)Q(B)-->Q(A)Q(B)(-) electron transfer suggests that conformational changes gating the latter process are distinct from those stabilizing the primary charge-separated state.

Effect of detergent concentration on the thermal stability of a membrane protein: The case study of bacterial reaction center solubilized by N,N-dimethyldodecylamine-N-oxide

We report on the response of reaction center (RC) from Rhodobacter sphaeroides (an archetype of membrane proteins) to the exposure at high temperature. The RCs have been solubilized in aqueous solution of the detergent N,N-dimethyldodecylamine-N-oxide (LDAO). Changes in the protein conformation have been probed by monitoring the variation in the absorbance of the bacteriochlorine cofactors and modification in the efficiency of energy transfer from tryptophans to cofactors and among the cofactors (through fluorescence measurements). The RC aggregation taking place at high temperature has been investigated by means of dynamic light scattering. Two experimental protocols have been used: (i) isothermal kinetics, in which the time evolution of RC after a sudden increase of the temperature is probed, and (ii) T-scans, in which the RCs are heated at constant rate. The analysis of the results coming from both the experiments indicates that the minimal kinetic scheme requires an equilibrium step and an irreversible process. The irreversible step is characterized by a activation energy of 205+/-14 kJ/mol and is independent from the detergent concentration. Since the temperature dependence of the aggregation rate was found to obey to the same law, the aggregation process is unfolding-limited. On the other hand, the equilibrium process between the native and a partially unfolded conformations was found to be strongly dependent on the detergent concentration. Increasing the LDAO content from 0.025 to 0.5 wt.% decreases the melting temperature from 49 to 42 degrees C. This corresponds to a sizeable (22 kJ/mol at 25 degrees C) destabilization of the native conformation induced by the detergent. The nature of the aggregates formed by the denatured RCs depends on the temperature. For temperature below 60 degrees C compact aggregates are formed while at 60 degrees C the clusters are less dense with a scaling relation between mass and size close to that expected for diffusion-limited aggregation. The aggregate final sizes formed at different temperatures indicate the presence of an even number of proteins suggesting that these clusters are formed by aggregation of dimers.

Cumulant Analysis of Charge Recombination Kinetics in Bacterial Reaction Centers Reconstituted into Lipid Vesicles

The kinetics of charge recombination between the primary photoxidized donor (P(+)) and the secondary reduced quinone acceptor (Q(B)(-)) have been studied in reaction centers (RCs) from the purple photosynthetic bacterium Rhodobacter sphaeroides incorporated into lecithin vesicles containing large ubiquinone pools over the temperature range 275 K </= T </= 307 K. To account for the non-exponential kinetics of P(+) re-reduction observed following a flash, a new approach has been developed, based on the following assumptions: 1) the exchange of quinone between different vesicles is negligible; 2) the exchange of quinone between the Q(B) site of the RC and the quinone pool within each single vesicle is faster than the return of the electron from the primary reduced acceptor Q(A)(-) to P(+); 3) the size polydispersity of proteoliposomes and the distribution of quinone molecules among them result in a quinone concentration distribution function, P(Q). The first and second moments of P(Q) have been evaluated from the size distribution of proteoliposomes probed by quasi-elastic light scattering (mean radius, = (50 +/- 15) nm). Following these premises, we describe the kinetics of P(+)Q(B)(-) recombination with a truncated cumulant expansion and relate it to P(Q) and to the free energy changes for Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer (DeltaG(AB)(o)) and for quinone binding (DeltaG(bind)(o)) at Q(B). The model accounts well for the temperature and quinone dependence of the charge recombination kinetics, yielding DeltaG(AB)(o) = -7.67 +/- 0.05 kJ mol(-1) and DeltaG(bind)(o) = -14.6 +/- 0.6 kJ mol(-1) at 298 K.