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Near-zero fouling monolayers based on binary patterned peptides allow low nanomolar detection of the matrix metalloproteinase-3 (MMP-3) directly in crude bovine serum, without sample pretreatment, secondary antibody detection or signal amplification. The peptide 3-MPA-HHHDD-OH (3-MPA, 3-mercaptopropionic acid) was found optimal compared to other binary patterned peptides based on 3-MPA-[A.sub.x]-[B.sub.y]-OH, where 0 [less than or equal to] x, y [less than or equal to] 5, and x + y = 5, and compared to PEG. In this study, amino acid A was His, Asp, Set, or Leu, and amino acid B was His, Asp, or Ser. Zwitterionic peptides and other peptides exhibited excellent resistance to nonspecific adsorption. Binary patterned peptides were capped with 3-MPA on the N-terminus providing a monolayer with the C-terminus carboxylic acid available to subsequently immobilize antibodies. Thereby, an IgG biosensor demonstrated the efficiency of binary patterned peptides in SPR biosensing with a detection limit of 1-10 pM in PBS, similar to other optical or electrochemical techniques. This protocol was applied to establish a calibration curve for MMP-3, an analyte of clinical interest for many pathologies and a potential indicator of cancer. The LOD for MMP-3 was 0.14 nM in PBS, with a linearity of up to 50 nM. With the use of PBS calibration, MMP-3 was quantified at low nanomolar in undiluted bovine serum. The SPR response in serum was statistically the same as in PBS. A sensor exposed to blank serum exhibited negligible nonspecific adsorption. Hence, binary patterned peptides are suitable for biosensing directly in complex biological matrixes. 10.1021/ac100035s

Short peptides, composed of polar or ionic amino acids, derived with a short organic thiol, significantly reduce nonspecific adsorption of proteins in complex biological matrices such as serum and crude cell lysate, which have nonspecific protein concentrations of 76 and 30-60 rag/ mL, respectively. Minimizing these nonspecific interaclions has allowed rapid and direct quantification of [beta]-lactamase in a crude cell lysate using a surface plasmon resonance (SPR) biosensor. A library of short peptides with varying chain length and amino acid composition were synthesized using a solid-phase approach. A 3-mercaptopropionic acid (3-MPA) linker was covalently attached to the amino terminus of the peptides to subsequentiy form a monolayer on gold in the form of 3-MPA-[(AA).sub.n]-OH, where n is the length of the amino acid chain (n = 2-5). Leu, Phe, Set, Asp, and His were selected to investigate the effect on nonspecific adsorption with different physicochemieal properties of the sidechains; aliphatic, aromatic, polar, acid, and base. Advancing contact angles measured the hydrophohicity of each peptldie self-assembled monolayer (SAM) and showed that hydrophilicity of the gold surface improved as the chain length of the polar or ionic peptides increased, while aromatic and aliphatie peptides decreased the hydrophilicity as the chain length increased. The nonspecific adsorption of undiluted bovine serum on SPR sensors prepared with the library of 3-MPA-[(AA).sub.n]-OH showed that the lowest nonspeciflc adsorption occurred with polar or ionic amino acids with a chain length of n = 5. We demonstrate that a monolayer composed of 3-MPA-[(Ser).sub.5]-OH has significant advantages, including the following: (1) it minimizes nonspecific adsorption in undiluted bovine serum; (2) it provides a high surface concentration of immobilized antibodies; (3) it shows a great retention of activity for the antibodies; (4) it improves the response from [beta]-inctamase by ~1 order of magnitude, compared to previous experiments; and (5) it allows direct quantification of submicromolar [beta]-lactamase concentration in a crude cell lysate with a nonspecific protein concentration of 30-60 mg/mL. The use of this peptide-hased monolayer offers great advantages for quantitative SPR biosensing in complex biological media.

The physiological application of amperometric adenosine triphosphate (ATP) microbiosensors for characterizing the stimulus-response at rat carotid bodies superfused with high potassium concentrations, during normoxic hypercapnia, and during hypoxia is demonstrated using the peripheral arterial chemoreceptors in the carotid body of rats as a model system. Amperometric microbiosensors based on glucose oxidase (GOD) and hexokinase (HEX) immobilized within a polymer matrix at the surface of Pt disk microelectrodes (diameter: 25 [micro]m) are positioned at a distance of approximately 100 [micro]m above the carotid body surface for detecting extracellular ATP. A linear calibration function of ATP microbiosensors in the physiologically relevant concentration range of 0-40 [micro]M ATP enables quantitative detection of ATP released at the carotid body surface in response to physiological stimuli. It is shown that these stimuli induce extracellular ATP release from the carotid body at levels of 4-10 [micro]M. Other electroactive neurotransmitters such as, e.g., catecholamines are coreleased by the carotid body at hypercapnic, hypoxic and high-potassium stimulus, are simultaneously detected utilizing a dual-electrode assembly with an ATP microbiosensor and a second bare channel providing a colocalized reference measurement for ATP quantification.

A general theoretical model for competitive dual-enzyme microbiosensors based on self-assembled monolayers (SAM) is presented. The model is derived for amperometric dual-enzyme ATP sensors and provides excellent agreement with experimental ATP measurements at 25 [micro]m diameter microelectrodes. In this model, the statistical probability of a glucose molecule in competition between two enzymes, glucose oxidase (GOD)/hexokinase (HEX), at the ATP sensor surface is combined with the enzymatic reaction rate. Thereby, a simple model predicting the sensor signal for varying surface concentrations of GOD and HEX, glucose concentration, and ATP concentration is obtained. Excellent agreement of the predicted current signal with experimentally obtained sensor signals was achieved at ATP concentrations between 10 and 300 [micro]M in a buffer containing glucose at physiologically relevant levels. Consequently, the development rime for new dual-enzyme biosensors can be reduced, and an analytical model for the sensor response function is provided facilitating the calibration of enzymatic biosensors.

Two mycocardial infarction biomarkers, myoglobin (MG) and cardiac troponin I (cTnI), were quantified at biological levels and in undiluted serum without sample pretreatment using surface plasmon resonance (SPR) sensors. To achieve detection of biomarkers in undiluted serum (72 mg/mL total protein concentration), minimization of the nonspecitic signal from the serum protein was achieved by immobilizing the antibody for the biomarkers on an N-hydroxysuccinimide activated 16-mercaptohexadecanoic acid self-assembled monolayer. This monolayer reduces the nonspecific signal from serum proteins in such a manner that short exposure of the sensor to serum prior to analysis prevents any further nonspecific adsorption during analysis. Thus, sensing of MG and cTnI was achieved on the basis of the difference between signals from the active sensor and a reference sensor that captured background interference. This resulted in direct measurement of these biomarkers in undiluted serum. Detection limits for both markers were below 1 ng/mL, which is below the threshold needed to detect myocardial infarction. Detecting biomarkers in the low ng/mL range without signal amplification in such a complex matrix as serum corresponds to a selectivity of [10.sup.8]. The root-mean-square-error (RMSE) of calibration was below 2 ng/mL.

Sensing of three cytokines related to chronic wound healing, interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-[alpha]), with detection limits at or below 1 ng/mL in buffered saline solution and spiked cell culture medium (CCM) has been achieved. Fiber-optic surface plasmon resonance (SPR) sensors are coated with an antibody binding layer and antibodies specific to the cytokine of interest are covalently attached to this layer. To achieve such detection limits in a complex medium such as CCM, total protein content of 4 mg/mL, the use of a novel N-hydroxysuccinimide ester of 16-mercaptohexadecanoic acid (NHS-MHA) is necessary. A comparison of the detection limits for IL-6 using currently widely used CM-dextran and NHS-MHA shows an improvement by a factor of 3 using NHS-MHA. The detection limits for the monitoring of cytokines in spiked saline solutions and CCM were similar for TNF-[alpha] and slightly higher for IL-1 and IL-6. The detection of each cytokine in the presence of interfering agents resulted in concentration prediction well within the error of calibration. The SPR sensors are stable in CCM after 20 min of pretreatment in CCM, minimizing the reliance on a reference sensor to quantify the cytokines in complex media. This technique enables a major advancement in the field of real-time monitoring of biologically relevant molecules in complex biological fluids.