Victor H. Perez-Luna

Rapid prototyping of patterned functional nanostructures

Living systems exhibit form and function on multiple length scales and at multiple locations. In order to mimic such natural structures, it is necessary to develop efficient strategies for assembling hierarchical materials. Conventional photolithography, although ubiquitous in the fabrication of microelectronics and microelectromechanical systems, is impractical for defining feature sizes below 0.1 micrometres and poorly suited to pattern chemical functionality. Recently, so-called ‘soft’ lithographic approaches have been combined with surfactant and particulate templating procedures to create materials with multiple levels of structural order. But the materials thus formed have been limited primarily to oxides with no specific functionality, and the associated processing times have ranged from hours to days. Here, using a self-assembling ‘ink’, we combine silica–surfactant self-assembly with three rapid printing procedures—pen lithography, ink-jet printing, and dip-coating of patterned self-assembled monolayers—to form functional, hierarchically organized structures in seconds. The rapid-prototyping procedures we describe are simple, employ readily available equipment, and provide a link between computer-aided design and self-assembled nanostructures. We expect that the ability to form arbitrary functional designs on arbitrary surfaces will be of practical importance for directly writing sensor arrays and fluidic or photonic systems.

A surface plasmon resonance array biosensor based on spectroscopic imaging

We have developed a multi-element transduction system which combines conventional SPR spectroscopy with one-dimensional SPR microscopy to create an effective platform for monitoring binding events on macro- or micro-patterned receptor arrays created on disposable sensor chips. This creates an effective platform for monitoring simultaneous binding events on each of the regions patterned with the receptors. This system has been specifically designed with commercially available components to allow relatively easy duplication. Furthermore, this system can use a proven, simple method to compensate for changes in the bulk index of refraction of the solution containing the analytes due to changes in temperature or solute concentration with simple modifications to the sensor chips alone. Preliminary results demonstrate how this system can be used to monitor several independent biospecific binding events simultaneously.

Surface plasmon resonance measurement of binding and dissociation of wild-type and mutant streptavidin on mixed biotin-containing alkylthiolate monolayers

Abstract The attachment of recognition elements to surfaces through streptavidin–biotin links has potential applications in sensor technology. Surface plasmon resonance (SPR) was used to monitor the kinetics of binding of streptavidin (SA) to mixed biotin-containing alkylthiolate monolayers (BTMs) on gold and the subsequent competitive desorption kinetics. Various binary compositions of BTMs were prepared from a mixture of biotinylated alkylthiol (BAT) and a poly(ethylene oxide) (PEO) alkylthiol. Differences in both the amount of adsorbed protein and the relative desorption rates between a low-binding affinity mutant (W120A) and wild-type streptavidin (WT) were observed as a function of the composition of the BTM. The rates of WT and W120A desorption from these mixed BTMs were correlated with the amount of biotin present on the surface. The results confirm the specific nature of the bonds which attach streptavidin to the biotinylated surface in the mixed BAT/PEO monolayer. However, when the PEO-terminated alkylthiolate was replaced with a simple methyl-terminated alkylthiolate, non-specific adsorption of the streptavidin dominates.

Fluorescence biosensing strategy based on energy transfer between fluorescently labeled receptors and a metallic surface

A new fluorescence-based biosensor is presented. The biosensing scheme is based on the fact that a fluorophore in close proximity to a metal film (<100 A) experiences strong quenching of fluorescence and a dramatic reduction in the lifetime of the excited state. By immobilizing the analyte of interest (or a structural analog of the analyte) to a metal surface and exposing it to a labeled receptor (e.g. antibody), the fluorescence of the labeled receptor becomes quenched upon binding because of the close proximity to the metal. Upon exposure to free analyte, the labeled receptor dissociates from the surface and diffuses into the bulk of the solution. This increases its separation from the metal and an increase of fluorescence intensity and/or lifetime of the excited state is observed that indicates the presence of the soluble analyte. By enclosing this system within a small volume with a semipermeable membrane, a reversible device is obtained. We demonstrate this scheme using a biotinylated self-assembled monolayer (SAM) on gold as our surface immobilized analyte analog, fluorescently labeled anti-biotin as a receptor, and a solution of biotin in PBS as a model analyte. This scheme could easily be extended to transduce a wide variety of protein-ligand interactions and other biorecognition phenomena (e.g. DNA hybridization) that result in changes in the architecture of surface immobilized biomolecules such that a change in the separation distance between fluorophores and the metal film is obtained.

SPR biosensors: simultaneously removing thermal and bulk-composition effects

Surface plasmon resonance (SPR) is an established method for sensing analytes by monitoring changes in the plasmon dispersion relation (PDR) at the interface of a thin metal film and a fluid. When SPR is used in sensors for specific analytes, the changes in the plasmon dispersion relation of interest are generated by the binding of analytes to receptors immobilized to the metal film. However, changes in the PDR can also be generated by changes in the index of refraction of the bulk solution containing the analytes via changes in composition or temperature. Thus, there exist inherent systematic errors in SPR based chemical sensing when temperature and/or concentration conditions are not carefully controlled. We have demonstrated the efficacy of a single, simple, and inexpensive method for simultaneously discriminating both effects from those of binding and/or debinding of analytes with a two-element SPR sensor array. Although two-element SPR arrays have been used before, their ability to simultaneously discriminate out both thermal and bulk-composition (TAB) effects (in both SPR spectroscopic and spectrophotometric schemes) has not been previously addressed. We show an example of a relatively inexpensive SPR biosensor instrument using a compensator element and its stability over a period of days. This demonstration has implications for the development of reliable SPR based chemical sensors for environmental and remote sensing applications.

Fluorescence lifetime-based phase-sensitive sensor array with LED or diode laser excitation for chemical and biosensing

Fluorescence lifetime-based sensors are well-suited for chemical and biological applications since they are relatively insensitive to background light intensity, fluctuations and bleaching of fluorophores. Examples of applications include biosensing strategies where binding or a target analyte to an immobilized biological receptor molecule results in a change in the fluorescence lifetime of a fluorescent reporter group. It is desirable in many instances to have a sensor array to monitor the simultaneous binding of several analytes. We have designed a multichannel system using LEDs (or laser diodes) to excite fluorescence, multiple photodetectors, and a multichannel computer algorithm-based phase meter. The multichannel phase meter utilizes a PC and multichannel digital acquisition board. The resolution of each channel in the multichannel phase meter has been estimated at approximately 0.05 degrees. The eigen-phase fluctuations for each channel of the system are approximately 0.15 degrees, which allow us to estimate the lifetime resolution as better than 10 ps. We estimate the processing time of phase measurements for each channel as less than 200 ms. The usefulness of the system has been demonstrated in several operational examples, including a multichannnel pH meter and a fluoresphore competitive immunoassay-based chemical sensor.

Compact LED-based phase fluorimeter detection system for chemical and biosensor arrays

This paper describes a design for an inexpensive phase fluorimeter based on blue or green light emitting diodes for use with chemical and biological sensors with fluorescence and phosphorescence lifetime-based transduction. The phase fluorimeter is based on a personal computer, two frequency synthesizers and off-the-shelf optical and electronic components and has an estimated lifetime resolution better than 10 ps. The phase fluorimeter is especially well-suited for implementation with arrays of chemically sensitive elements. The data acquisition system allows rapid monitoring of light emission from fluors or phosphors immobilized in the chemically sensitive array elements, each of which can be designed to be responsive to a particular chemical analyte. This paper describes chemically sensitive elements based on ultrathin films of porous polymers and on self-assembled monolayers. The paper focuses on methods for detection of low levels of luminescence emission from micropatterned array elements that comprise sensor arrays. Of particular importance is the detection of low levels of fluorescence and phosphorescence from SAMs of alkanethiolates on thin gold and silver films. Methods for enhancing the luminescence yield from these SAMs include optimization of the dielectric environment of the luminescent dyes and surface plasmon resonance enhanced excitation.

Surface plasmon resonance based array biosensors for multianalyte detection

Surface plasmon resonance (SPR) is a phenomenon wherein the reflectance versus angle-of-incidence profile for a thin gold film illuminated with p-polarized light has a distinct minimum at a particular angle. This minimum of reflectance is due to an absorption of the light energy by the surface electron plasma of the metal occurring when the surface- parallel components of the light and plasmon propagation vectors match up. The value of this particular angle of incidence changes in proportion to the degree of adsorption of analytes to the metal film. This allows SPR to be used as a simple, noninvasive, optical tool for measuring the binding of chemical analytes. With a predetermined pattern of chemically specific receptors bound to the gold film, it is possible to detect a variety of species and concentrations of analytes, provided that one has a sensor platform capable of resolving the different reactions in each element of the receptor array. We have developed such a platform which is capable of optically monitoring an array of analyte receptors immobilized on gold coated microscope slides in real time. Moreover, the optical resolution of sensor platform allows the receptors to be micro-patterned.

Compact phase-sensitive multichannel detection system with array measurements of biosensor chips

In many medical, biological, chemical, and environmental applications it is desirable not only to monitor one specific chemical or biological species, but several simultaneously. Thus, we have focused our efforts on development of a detection system for multi-analyte sensor arrays that is able to monitor the changes in fluorophore lifetimes (via the detection of phase shifts) corresponding to the presence of many analytes of interest in near-real time. We describe a phase-sensitive electronic detection system employing a multianode photomultiplier tube. This system utilizes the frequency-domain method of time-resolved spectroscopy and is also suitable for lifetime-based imaging.