We examine through analytical calculations and finite element simulations how the detection efficiency of disk and wire-like biosensors in unmixed fluids varies with size from the micrometer to nanometer scales. Specifically, we determine the total flux of DNA-like analyte molecules on a sensor as a function of time and flow rate for a sensor incorporated into a microfluidic system. In all cases, sensor size and shape profoundly affect the total analyte flux. The calculations reveal that reported femtomolar detection limits for biomolecular assays are very likely an analyte transport limitation, not a signal transduction limitation. We conclude that without directed transport of biomolecules, individual nanoscale sensors will be limited to picomolar-order sensitivity for practical time scales.Tremendous progress is being made in the development of microanalytical systems for biosensing, driven by parallel advances in biotechnology, microtechnology, and microfluidics. 1,2 The advantages of small, highly integrated systems include more rapid and multiplexed analysis and reagent sample volumes reduced to the microliter range. When combined with innovative signal transduction technology, microsystems have recently achieved specific biomolecular detection at roughly femtomolar (fM) concentrations, corresponding to just a few thousand (or even a few hundred) analyte molecules in the sample volume. [3][4][5] Concurrently, many research groups have been developing micrometer or nanometer scale sensing elements based on novel transduction mechanisms. [5][6][7][8][9][10][11] Many researchers of nanometer-scale phenomena focus on the fact that miniaturizing a sensor often increases its signal-to-noise ratio (S:N), an inherent advantage for signal transduction, but the effect of nanoscale miniaturization on the overall sensitivity, which includes mass transport effects, has not been widely considered. For example, whether nanometer-scale sensors are intrinsically more sensitive overall than micrometer-scale sensors has not been fully examined. In this letter, we use experimentally verified [12][13][14] analytical solutions to examine the maximum sensitivity with which micro-to-nanoscale sensors of various geometries can detect biomolecules from solution. Our principal goal is to explicitly examine mass transport effects on biosensing at the nanoscale; however, the calculations also lead us to conclude that reported femtomolar detection limits for bioassays are likely an analyte transport limitation, not a signal transduction limitation. The implication is that, without methods to actively direct biomolecules to a sensor surface, individual nanoscale sensors will be subject to picomolarorder detection limits for practical time scales. 15 In the past decade, several papers have analyzed the effect of flow, 16-18 size, 13 or adsorption isotherms 19 on biomolecular adsorption; however, none have explicitly examined the effect at nanometer length scales. This effect is most easily examined using a simple geometrysa singl...

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Abstract:We describe the complementary use of X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopy to quantitatively characterize the immobilization of thiolated (dT)25 single-stranded DNA (ssDNA) on gold. When electron attenuation effects are accurately accounted for in the XPS analysis, the relative coverage values obtained by the two methods are in excellent agreement, and the absolute coverage can be calculated on the basis of the XPS data. The evolution of chemically specific spectral signatures during immobilization indicates that at lower coverages much of the DNA lies flat on the surface, with a substantial fraction of the thymine bases chemisorbed. At higher immobilization densities, the (dT) 25 film consists of randomly coiled ssDNA molecules each anchored via the thiol group and at possibly one or two other bases. We use two examples to demonstrate how the quantitative analysis can be applied to practical problems: the effects of different buffer salts on the immobilization efficiency; the immobilization kinetics. Buffers with divalent salts dramatically increase the efficiency of immobilization and result in very high surface densities (>5 × 10 13 / cm 2 ), densities that may only be possible if the divalent counterions induce strong attractive intermolecular interactions. In contrast with previous reports of alkanethiol adsorption kinetics on gold, ssDNA immobilization in 1 M phosphate buffer does not occur with Langmuir kinetics, a result attributable to rearrangement within the film that follows the initial adsorption.

We describe the use of self-assembled films of thiolated (dT)25 single-stranded DNA (ssDNA) on gold as a model system for quantitative characterization of DNA films by X-ray photoelectron spectroscopy (XPS). We evaluate the applicability of a uniform and homogeneous overlayer-substrate model for data analysis, examine model parameters used to describe DNA films (e.g., density and electron attenuation length), and validate the results. The model is used to obtain quantitative composition and coverage information as a function of immobilization time. We find that when the electron attenuation effects are properly included in the XPS data analysis, excellent agreement is obtained with Fourier transform infrared (FTIR) measurements for relative values of the DNA coverage, and the calculated absolute coverage is consistent with a previous radiolabeling study. Based on the effectiveness of the analysis procedure for model (dT)25 ssDNA films, it should be generally valid for direct quantitative comparison of DNA films prepared under widely varying conditions.

We describe self-assembly of ssDNA brushes that exploits the intrinsic affinity of adenine nucleotides (dA) for gold surfaces. The grafting density and conformation of these brushes is deterministically controlled by the length of the anchoring dA sequences, even in the presence of thymine nucleotides (dT) FTIR ͉ gold ͉ immobilization ͉ oligonucleotides ͉ x-ray photoelectron spectroscopy T he properties of surfaces functionalized with ssDNA exhibit a remarkable richness that underlies the versatility of these surfaces in a wide range of applications. The ssDNA brushes described in this work offer unique properties for two types of applications: control of nanoscale self-assembly (1, 2) and design and operation of biosensors (3-8). In general, a critical attribute of a ssDNA-modified surface is efficient and reproducible hybridization with target DNA. Model studies using thiolated DNA probes have shown that efficient hybridization occurs when the spacing between immobilized DNA probes is large and the orientation of the probes is upright (4-11). Unfortunately, reproducible preparation of DNA films possessing both of these qualities remains challenging (12-14). For example, it is generally observed that when the grafting density is low (Ͻ10 13 cm Ϫ2 ), i.e., the spacing between probes is comparable to their length, DNA immobilizes in a flat conformation through nonspecific adsorption (9,15,16). This observation can be largely explained by conventional polyelectrolyte brush theory, which predicts that negatively charged DNA strands should only assume upright conformations in densely packed films, where repulsive interactions force the strands to extend away from the surface (7)..Surface passivation is a common strategy used to decouple the DNA conformation from grafting density. Passivation prevents nonspecific interactions between the surface and DNA or other biomolecules, which enables widely spaced DNA probes to maintain an upright orientation. In a common implementation of this strategy, films of thiol-anchored DNA can be exposed to a solution containing a competing molecule, such as mercaptohexanol (MCH), which displaces most of the DNA from the surface and forces the remaining DNA strands into an upright conformation (10,(17)(18)(19). Alternatively, grafting densities can be adjusted by coupling functionalized DNA to a bifunctional self-assembled monolayer (6).Our objective in this study is to control independently and deterministically both the conformation and grafting density of DNA. We realize this objective by introducing anchoring sequences of adenine nucleotides (dA), which in our previous work have been shown to have a high intrinsic affinity for gold surfaces (20). Here, the function of the adenine blocks [(dA)] extends, however, beyond simple anchoring: They preferentially bind to gold surfaces and block nearly all of the adsorption sites, preventing nonspecific binding of other sequences to these surfaces (an effect similar to that of using the MCH posttreatment or a bifunctional self-assembled mon...

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The radii of octadecanethiol spots deposited by an atomic force microscope tip onto a gold surface were studied as a function of contact time and humidity. The deposition is well described by twodimensional diffusion from an annular source of constant concentration, with a surface diffusion coefficient of 8400 nm 2 s 21 , independent of humidity. Facile transfer is observed even after near continuous deposition for more than 24 h in a dry N 2 environment, indicating that a water meniscus is not required. DOI: 10.1103/PhysRevLett.88.156104 PACS numbers: 68.37.Ps, 68.43.Jk, 81.16.Nd, 81.16.Rf Since the first use of alkanethiols by Nuzzo and Allara [1] to form a self-assembled monolayer (SAM) on a gold surface, the number of applications for thiolated hydrocarbons has increased rapidly [2], ranging from resists for electronics to biosensor substrates. The major advantages offered by thiol SAMs include long-term stability, versatility in terminal functionality, and ease of use. Perhaps the most attractive feature is their utility for forming small, reproducible surface features outside of a clean room. For example, facile creation of micron-scale patterns has been demonstrated by "stamping" thiols onto a surface using a flexible polymer master, a technique known as microcontact printing (mCP) [3]. Even smaller, nanometer-scale features can be patterned from thiols using the recently developed "Dip Pen Nanolithography" (DPN) [4], where alkanethiols are "written" via transfer from an atomic force microscope (AFM) tip to a surface. The prospect of nanometer-scale control over physical dimensions combined with the flexible terminal group chemistry makes thiol patterning a critical component of many proposed nanotechnologies.Because alkanethiol patterning is emerging as a key technique for nanofabrication, it is crucial that the mechanisms of deposition -and thereby the ultimate technological potential-be understood. The most important process of the deposition may be diffusion, which controls both the extent and the quality of an alkanethiol SAM. As previously noted in a study of mCP [5], the spatial resolution of a pattern is fundamentally limited by diffusion of the thiol "ink." First, diffusion of the thiol beyond the initial contact area causes the pattern to be wider than the stamp (for mCP) or the AFM tip terminus (for DPN). Although there have been a few attempts to measure the diffusion rate of thiols on gold [5][6][7][8], these measurements have been mostly phenomenological and thus have not reported diffusion coefficients. A second, more subtle, effect of diffusion is its role in the phase transition that occurs during SAM deposition: At a critical surface concentration, the adsorbed thiols reorient from a prone to a standing orientation. This change in orientation affects physical properties of the SAM, such as friction, as well as important chemical properties, such as its ability to mask the substrate from an etchant [9].Another important issue in determining the overall potential of DPN is the role of ...

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