Plasmonic Sensing on Symmetric Nanohole Arrays Supporting High-Q Hybrid Modes and Reflection Geometry.

Refractometric sensors utilizing surface plasmon resonance (SPR) should satisfy a series of performance metrics, bulk sensitivity, thin-film sensitivity, refractive-index resolution, and high-Q-factor resonance, as well as practical requirements such as manufacturability and the ability to separate optical and fluidic paths via reflection-mode sensing. While many geometries such as nanohole, nanoslit, and nanoparticles have been employed, it is nontrivial to engineer nanostructures to satisfy all of the aforementioned requirements. We combine gold nanohole arrays with a water-index-matched Cytop film to demonstrate reflection-mode, high-Q-factor (Qexp = 143) symmetric plasmonic sensor architecture. Using template stripping with a Cytop film, we can replicate a large number of index-symmetric nanohole arrays, which support sharp plasmonic resonances that can be probed by light reflected from their backside with a high extinction amplitude. The reflection geometry separates the optical and microfluidic paths without sacrificing sensor performance as is the case of standard (index-asymmetric) nanohole arrays. Furthermore, plasmon hybridization caused by the array refractive-index symmetry enables dual-mode detection that allows distinction of refractive-index changes occurring at different distances from the surface, making it possible to identify SPR response from differently sized particles or to distinguish binding events near the surface from bulk index changes. Due to the unique combination of a dual-mode reflection-configuration sensing, high-Q plasmonic modes, and template-stripping nanofabrication, this platform can extend the utility of nanohole SPR for sensing applications involving biomolecules, polymers, nanovesicles, and biomembranes.

Purity and stability of the membrane-limited glucocorticoid receptor agonist dexamethasone-BSA.

Cellular effects of glucocorticoids can be separated into classical transcriptional regulation via activation of the canonical nuclear glucocorticoid receptor and rapid actions mediated by activation of one or more putative membrane-associated glucocorticoid receptors that regulate both transcriptional and non-transcriptional signaling. Dexamethasone-bovine serum albumin (Dex-BSA) is one of several membrane-limited steroid receptor agonists. Dex-BSA and other steroid conjugates such as corticosterone-, estradiol- and testosterone-BSA have been used to study rapid steroid effects initiated by putative membrane receptors. The purity and stability of the steroid-BSA conjugate is crucial, therefore, since any steroid that is not bound to or that dissociates from the BSA conjugate could penetrate into the intracellular compartment and confound the experiment. We used fluorine NMR to determine if free Dex could be detected in a commercially available Dex-BSA dissolved in H2O. Non-covalently bound Dex was detected in the Dex-BSA solution, but the level of free Dex remained constant over time and with increasing temperature, indicating that the free Dex was not a result of instability of the Dex-BSA conjugate. The free Dex was lost when the Dex-BSA was denatured and subjected to dialysis, which suggested that it was trapped in the Dex-BSA three-dimensional structure and not covalently bound to the BSA. The purified, renatured Dex-BSA retained its rapid activity, which confirmed that the observed effects of Dex-BSA are not caused by non-covalently-bound Dex. Therefore, the Dex contaminant found in the Dex-BSA solution is likely to be tightly, but non-covalently, bound to BSA, and the Dex-BSA activity remains membrane-limited. Our findings indicate that Dex-BSA remains a suitable membrane-restricted glucocorticoid receptor agonist, but suggest that denaturing purification is a useful control for the study of membrane-initiated steroid-BSA actions.

Mineral supplementation stimulates the immune system and antioxidant responses of dairy cows and reduces somatic cell counts in milk.

The aim of this study was to evaluate whether the use of subcutaneous mineral supplementation would affect metabolic parameters, immunological response, milk quality and composition of dairy cows in the postpartum period. Twelve pregnant primiparous Holstein cows, were divided into two groups: six animals supplemented with the mineral complex (magnesium, phosphorus, potassium, selenium and copper), and six animals used as controls. Milk samples were collected every two other weeks postpartum up to sixty days of lactation to analyze composition and quality. Blood samples were collected, and the levels of ketone bodies, total proteins, glucose, albumin, and globulin were measured. The catalase and superoxide enzymes, reactive oxygen species, tumor necrosis factor, and interleukins were determined. Animals supplemented with minerals showed lower levels of ketone bodies and somatic cell counts on days 30, 45 and 60 of the experiment, without changes in milk composition compared to the control group. Supplemented cows had lower levels reactive oxygen species and increased superoxide enzymes activity. Total protein, globulin and cytokine levels were higher in cows supplemented with mineral complexes. Therefore, we can conclude that subcutaneous mineral supplementation improved the immune response and minimized the oxidative stress in dairy cows during lactation.

Development of Polyclonal Antibodies for Detection of Diosbulbin B-Derived cis-Enedial Protein Adducts.

Diosbulbin B (DSB), a major component of herbal medicine Dioscorea bulbifera L. (DB), can be metabolized to an electrophilic intermediate, DSB-derived cis-enedial (DDE). DDE was suggested to contribute to the hepatotoxicity observed in experimental animals and humans after their exposure to DSB. Our previous work found that DDE reacted with primary amino and/or sulfhydryl groups of hepatic protein. The objective of the study was to develop polyclonal antibodies that can recognize DDE-derived protein adducts. Immunogens synthesized from DDE and keyhole limpet hemocyanin were employed to raise polyclonal antibodies in rabbits. An enzyme-linked immunosorbent assay (ELISA) demonstrated high titers of antisera obtained from immunized rabbits. Immunoblot analysis showed that DDE-modified bovine serum albumin (BSA) was recognized by the obtained polyclonal antibodies in a concentration-dependent manner and without cross-reaction to native BSA. Competitive ELISA and competitive immunoblot analyses defined the specificity of the antibodies to recognize BSA modified by DDE. Immunoblot analysis also detected a multitude of chemiluminescent bands with a variety of molecular weights in liver homogenates that were harvested from mice treated with DSB. In summary, we have successfully raised polyclonal antibodies to detect protein adducts derived from DDE.

Proteomic studies with a novel nano-magnetic chelating system to capture metalloproteins and its application in the preliminary study of monocyte and macrophage sub-secretome.

A new chelating chromatography method was developed based in the use of magnetic iron oxide nanoparticles functionalized with EDTA-TMS ((N-(trimethoxysilylpropyl)ethylenediaminetriacetate trisodium salt). These particles combine a high surface area, biocompatibility and magnetic removal from solution, with the chelating affinity towards metal ions. The particles were used to selectively capture metallo-dependant proteins in secretome obtained from human monocytes and mouse macrophages. Secreted metallo-dependant proteins are highly important sources of information since they are involved in several pathological processes. The identification of secreted proteins involved in these processes is highly important for diagnosis or monitoring the progression of a disease. In this multiple-approach study it was possible to not only selectively capture several secreted metallo-dependant proteins, but also to significantly avoid masking proteins such as the highly abundant albumin form the fetal bovine serum used to supplement the cell culture medium. Overall, the magnetic nanoparticle-based chelating chromatography method developed here has proved to be a sensitive, low cost, and a quick tool for sample treatment in order to selectively enrich metalloproteins while overcoming the contamination of highly abundant proteins.

Mechanisms of Antigen Adsorption Onto an Aluminum-Hydroxide Adjuvant Evaluated by High-Throughput Screening.

The adsorption mechanism of antigen on aluminum adjuvant can affect antigen elution at the injection site and hence the immune response. Our aim was to evaluate adsorption onto aluminum hydroxide (AH) by ligand exchange and electrostatic interactions of model proteins and antigens, bovine serum albumin (BSA), ß-casein, ovalbumin (OVA), hepatitis B surface antigen, and tetanus toxin (TT). A high-throughput screening platform was developed to measure adsorption isotherms in the presence of electrolytes and ligand exchange by a fluorescence-spectroscopy method that detects the catalysis of 6,8-difluoro-4-methylumbelliferyl phosphate by free hydroxyl groups on AH. BSA adsorption depended on predominant electrostatic interactions. Ligand exchange contributes to the adsorption of ß-casein, OVA, hepatitis B surface antigen, and TT onto AH. Based on relative surface phosphophilicity and adsorption isotherms in the presence of phosphate and fluoride, the capacities of the proteins to interact with AH by ligand exchange followed the trend: OVA < ß-casein < BSA < TT. This could be explained by both the content of ligands available in the protein structure for ligand exchange and the antigen's molecular weight. The high-throughput screening platform can be used to better understand the contributions of ligand exchange and electrostatic attractions governing the interactions between an antigen adsorbed onto aluminum-containing adjuvant.

Quarter variation and correlations of colostrum albumin, immunoglobulin G1 and G2 in dairy cows.

A high variation in immunoglobulin G1 (IgG1) concentration in first milked quarter colostrum has been reported, but BSA quarter colostrum variation is not known. The occurrence of serum albumin in milk has been attributed to increased blood-milk barrier penetration. Reports of serum albumin binding to the Fc Receptor of the neonate, the receptor thought to be responsible for IgG1 transcytosis, suggested that a correlation with the appearance of IgG1 in colostrum of dairy cows was likely. The objective of the study was to establish the quarter colostrum concentration and mass of immunoglobulins and serum albumin. First colostrum was quarter collected within 4 h of parturition from healthy udders of 31 multiparous dairy cows. Individual quarter colostrum weight was determined and a sample of each was frozen for subsequent analysis. Concentrations of immunoglobulin G1, G2, and BSA were measured by ELISA and total mass of components was calculated. In addition, colostrum was also analysed for L-lactate dehydrogenase activity. Analysis of concentration and mass of BSA, immunoglobulin G1, G2 established that the quarter variations were different by cow, quarter and quarter within cow. Partial correlations corrected for colostrum weight indicated that BSA and IgG2 concentration and mass are closely correlated while that of BSA and IgG1 concentration and mass exhibited no correlation suggesting that BSA and IgG1 may have different transport mechanisms. Interestingly, immunoglobulin G1 and G2 concentration and mass exhibited strong correlations suggesting that also some unknown mechanism of immunoglobulin G2 appearance in colostrum is occurring. Finally, no measured protein exhibited any correlation with the activity of lactate dehydrogenase in colostrum.

Development of continuous microwave-assisted protein digestion with immobilized enzyme.

In this study, an easy and efficiency protein digestion method called continuous microwave-assisted protein digestion (cMAED) with immobilized enzyme was developed and applied for proteome analysis by LC-MS(n). Continuous microwave power outputting was specially designed and applied. Trypsin and bromelain were immobilized onto magnetic micropheres. To evaluate the method of cMAED, bovine serum albumin (BSA) and protein extracted from ginkgo nuts were used as model and real protein sample to verify the digestion efficiency of cMAED. Several conditions including continuous microwave power, the ratio of immobilized trypsin/BSA were optimized according to the analysis of peptide fragments by Tricine SDS-PAGE and LC-MS(n). Subsequently, the ginkgo protein was digested with the protocols of cMAED, MAED and conventional heating enzymatic digestion (HED) respectively and the LC-MS(n) profiles of the hydrolysate was compared. Results showed that cMAED combined with immobilized enzyme was a fast and efficient digestion method for protein digestion and microwave power tentatively affected the peptide producing. The cMAED method will be expanded for large-scale preparation of bioactive peptides and peptide analysis in biological and clinical research.

Body surface area predicts plasma oxaliplatin and pharmacokinetic advantage in hyperthermic intraoperative intraperitoneal chemotherapy.

BACKGROUND: Hyperthermic intraoperative intraperitoneal chemotherapy (HIPEC) is used to treat peritoneal surface-spreading malignancies to maximize local drug concentrations while minimizing systemic effects. The pharmacokinetic advantage of HIPEC is defined as the intraperitoneal to intravascular ratio of drug concentrations. We hypothesized that body surface area (BSA) would correlate with the pharmacokinetic advantage of HIPEC. Because oxaliplatin is administered in 5 % dextrose, we hypothesized that BSA would correlate with glycemia. METHODS: We collected blood and peritoneal perfusate samples from ten patients undergoing HIPEC with a BSA-based dose of 250 mg/m(2) oxaliplatin, and measured drug concentrations by inductively coupled plasma mass spectrophotometry. We monitored blood glucose for 24 h postoperatively. Areas under concentration-time curves (AUC) were calculated by trapezoidal rule. Pharmacokinetic advantage was calculated by (AUC[peritoneal fluid]/AUC[plasma]). We used linear regression to test for statistical significance. RESULTS: Higher BSA was associated with lower plasma oxaliplatin AUC (p = 0.0075) and with a greater pharmacokinetic advantage (p = 0.0198) over the 60-minute duration of HIPEC. No statistically significant relationships were found between BSA and blood glucose AUC or peak blood glucose levels. CONCLUSIONS: Higher BSA is correlated with lower plasma drug levels and greater pharmacokinetic advantage in HIPEC, likely because of increased circulating blood volume with inadequate time for equilibration. Plasma glucose levels after oxaliplatin HIPEC were not clearly related to BSA.

Model immunoassay on silicon surfaces: vertical and lateral nanostructure vs. protein coverage.

To provide complete characterization of immunoassay on silicon biosensor surfaces, atomic force microscopy, (angle-resolved) X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry were applied to examine Si(3)N(4) surfaces modified with (3-aminopropyl)triethoxysilane, coated with gamma globulins (IgG), blocked with bovine serum albumin and then reacted with anti-IgG antibody for two complementary pairs (rabbit and mouse IgG) at various concentrations (from 0.3 nM to 330 nM). Protein coverage, as reflected in (amine to total N1s) XPS signal ratio and determined from ARXPS, decreases slightly due to blocking and then increases monotonically for anti-IgG antibody concentrations higher than 1 nM. AFM images reveal hardly any change of lateral nanostructure due to blocking but response to antibody solutions, based on both the mean size (from autocorrelation) and dominant spacing (from Fourier analysis) of surface features, similar to that given by ARXPS. AFM height histograms provided information about the vertical nanostructure and the parameters of height distribution (average height, spread - roughness and skewness) were distinctly influenced by coating, blocking and immunoreaction. Average protein layer thickness values determined based on protein structure (molecular weight, dimensions) and surface coverage provided from ARXPS were in accord with average height of protein layer determined from AFM. TOF-SIMS analysis indicated that BSA blocks free surface sites and in addition replaces some already adsorbed IgGs.

Cow's milk proteins in human milk.

Cow's milk proteins (CMPs) are among the best characterized food allergens. Cow's milk contains more than twenty five different proteins, but only whey proteins alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin (BSA), and lactoferrin, as well as the four caseins, have been identified as allergens. Aim of this study was to investigate by proteomics techniques cow's milk allergens in human colostrum of term and preterm newborns' mothers, not previously detected, in order to understand if such allergens could be cause of sensitization during lactation. Term colostrum samples from 62 healthy mothers and preterm colostrum samples from 11 healthy mothers were collected for this purpose. The most relevant finding was the detection of the intact bovine alpha-S1-casein in both term and preterm colostrum. Using this method, which allows direct proteins identification, beta-lactoglobulin was not detected in any of colostrum samples. According to our results bovine alpha 1 casein that is considered a major cow's milk allergen is readily secreted in human milk: further investigations are needed in order to clarify if alpha-1-casein has a major role in sensitization or tolerance to cow's milk of exclusively breastfed predisposed infants.

Fluorescence detection of adenosine triphosphate through an aptamer-molecular beacon multiple probe.

An aptamer-molecular beacon (MB) multiple fluorescent probe for adenosine triphosphate (ATP) assay is proposed in this article. The ATP aptamer was used as a molecular recognition part, and an oligonucleotide (short strand, SS) partially complementary with the aptamer and an MB was used as the other part. In the presence of ATP, the aptamer bound with it, accompanied by the hybridization of MB and SS and the fluorescence recovering. Wherever there is only very weak fluorescence can be measured in the absence of ATP. Based on the relationship of recovering fluorescence and the concentration of ATP, a method for quantifying ATP has been developed. The fluorescence intensity was proportional to the concentration of ATP in the range of 10 to 500 nM with a detection limit of 0.1 nM. Moreover, this method was able to detect ATP with high selectivity in the presence of guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP). This method is proved to be simple with high sensitivity, selectivity, and specificity.

Surface-enhanced Raman scattering using silver nanocluster on anodic aluminum oxide template sensor toward protein detection.

The affordable surface-enhanced Raman scattering (SERS) substrates, with a structure consisting of densely distributed round-shape silver nanoclusters on anodic aluminum oxide (AAO) template, is fabricated by magnetron sputtering and anodization processes. The physical investigations show that the silver nanoclusters with size distribution ranging from 10 to 30 nm uniformly distributed on the top and in the bottom of the AAO nanochannels. The SERS activities from adsorbed probe molecules, i.e., methylene blue, on the SERS substrate surface indicate a high Raman enhancement factor for trace organic analysis. The SERS substrate is successfully utilized in the detection of a trace amount of three different proteins, bovin serum albumin, immunoglobulin G, and cardiac troponin T, also adsorbed on the substrate surface. Several spectral bands containing important molecular structures of these proteins are clearly observed and identified. The obtained results indicated a step forward to label-free biomolecular detections in chip-based biosensors.

Antigen heterologous enzyme linked immunosorbent assay for the measurement of DHEA in serum.

Homologous and heterologous combinations of enzyme conjugate and antibody in steroid enzyme immunoassay (EIA) influences unlabeled steroid recognition by antibody that affects sensitivity of the assay. To develop dehydroepiandrosterone (DHEA) antigen heterologous enzyme linked immunosorbent assay (ELISA), antibodies were generated against DHEA-3-hemisuccinate-bovine serum albumin (DHEA-3-HS-BSA), DHEA-7-carboxymethyloxime-bovine serum albumin (DHEA-7-CMO-BSA), and DHEA-17-carboxymethyloxime-bovine serum albumin (DHEA-17-CMO-BSA). Five horseradish peroxidase (HRP) enzyme conjugates were prepared using five testosterone derivatives [testosterone-3-CMO (T-3-CMO), testosterone-17-HS (T-17-HS), testosterone-17-glucuronoside (T-17-G), testosterone-19-carboxymethylether (T-19-CME), and testosterone-11-HS (T-11-HS)]. Fifteen antigen heterologous combinations of antibody and enzyme conjugates were evaluated in the standard binding assay; only two combinations showed binding. The use of antigen heterologous combination (different antigen in label than the immunogen) resulted in development of a simple, direct, and convenient assay as it permits the direct addition of the serum sample into the assay and it requires only 1.5 h to complete.

Modified fast procedure for the detection and screening of antiglycative phytochemicals.

In an attempt to elevate temperature to facilitate glycation, a nonenzymatic reaction by incubation of bovine serum albumin (BSA) and fructose at 50 °C for 24 h has been developed. As conducted and compared to a routine procedure by incubation of BSA and fructose at 37 °C for 168 h, the reactant fluorescence intensities and SDS-PAGE-detected glycated BSA quantities produced by both test temperatures increased with time of incubation. As the Amadori products and α-dicarbonyl compounds during incubation were quantified, both quantities produced at each temperature also increased with an increase of time of incubation, and their trends of changes at both temperatures were similar. In practical application for the detection and screening of the antiglycative phytochemicals, each of 20 peanut root extracts was introduced to a series of BSA-fructose solutions and incubated at 37 and 50 °C for 168 and 24 h, correspondingly. All extracts exhibited notable activities and varied depending on peanut origins. Pair comparison of the resultant antiglycative activities determined at 37 and 50 °C showed that both determined activities for each peanut root extract deviated limitedly. As further analyzed, SDS-PAGE-detected glycated BSA quantities formed at 50 °C were closely proportional to the antiglycative activities determined on the basis of their fluorescence intensities. It is of merit to demonstrate that fluorescence-based determination of BSA-fructose reactant after incubation at 50 °C for 24 h is practical and time-saving in the detection and screening of antiglycative phytochemicals.

Confident identification of 3-nitrotyrosine modifications in mass spectral data across multiple mass spectrometry platforms.

3-nitrotyrosine (3NT) is an oxidative posttranslational modification associated with many diseases. Determining the specific sites of this modification remains a challenge due to the low stoichiometry of 3NT modifications in biological samples. Mass spectrometry-based proteomics is a powerful tool for identifying 3NT modifications, however several reports identifying 3NT sites were later demonstrated to be incorrect, highlighting that both the accuracy and efficiency of these workflows need improvement. To advance our understanding of the chromatographic and spectral properties of 3NT-containing peptides we have adapted a straightforward, reproducible procedure to generate a large set of 3NT peptides by chemical nitration of a defined, commercially available 48 protein mixture. Using two complementary LC-MS/MS platforms, a QTOF (QSTAR Elite) and dual pressure ion trap mass spectrometer (LTQ Velos), we detected over 200 validated 3NT-containing peptides with significant overlap in the peptides detected by both systems. We investigated the LC-MS/MS properties for each peptide manually using defined criteria and then assessed their utility to confirm that the peptide was 3NT modified. This broad set of validated 3NT-containing peptides can be utilized to optimize mass spectrometric instrumentation and data mining strategies or further develop 3NT peptide enrichment strategies for this biologically important, oxidative posttranslational modification.

Two-dimensional strong cation-exchange liquid chromatography/reversed-phase pressurized capillary electrochromatography for separation of complex samples.

A 2-D separation platform was constructed using micro strong cation-exchange liquid chromatography (µ-SCXLC) and reversed-phase pressurized capillary electrochromatography (RP-pCEC) for the analysis of complex samples. Samples were fractionated by the first-dimension µ-SCXLC with a linear solvent gradient and then injected into the second-dimension RP-pCEC for further separation. The µ-SCXLC/RP-pCEC 2-D system with three separation mechanisms, namely strong cation-exchange, reversed-phase chromatography and electrophoresis, provided high selectivity, high resolution and high peak capacity compared to one-dimensional chromatographic approaches. Separation effectiveness of this 2-D system was demonstrated by the analysis of different kinds of complex samples, such as traditional Chinese medicine Cortex Phellodendri, bovine serum albumin (BSA) tryptic digest and real serum tryptic digest. A theoretical peak capacity of approximately 1200 was achieved, which proves its promising potential for the separation and analysis of complex samples.

Nanoporous alumina-based interferometric transducers ennobled.

A high fidelity interferometric transducer is designed based on platinum-coated nanoporous alumina films. The ultrathin metal coating significantly improves fidelity of the interferometric fringe patterns in aqueous solution and increases the signal-to-noise ratio. The performance of this transducer is tested with respect to refractive index unit (RIU) sensitivity measured as a change in effective optical thickness (EOT) in response to a solvent change and compared to porous silicon based transducers. RIU sensitivity in the order of 55% is attainable for porous alumina providing excellent signal-to-noise ratio, which exceeds the sensitivity of current interferometric transducers. Finally, as a proof-of-principle, we demonstrate biosensing with two distinct immunoglobulin antibodies.

One-step method for generating PEG-like plasma polymer gradients: chemical characterization and analysis of protein interactions.

In this work we report a one-step method for the fabrication of poly(ethylene glycol) PEG-like chemical gradients, which were deposited via continuous wave radio frequency glow discharge plasma polymerization of diethylene glycol dimethyl ether (DG). A knife edge top electrode was used to produce the gradient coatings at plasma load powers of 5 and 30 W. The chemistry across the gradients was analyzed using a number of complementary techniques including spatially resolved synchrotron source grazing incidence FTIR microspectroscopy, X-ray photoelectron spectroscopy (XPS) and synchrotron source near edge X-ray absorption fine structure (NEXAFS) spectroscopy. Gradients deposited at lower load power retained a higher degree of monomer like functionality as did the central region directly underneath the knife edge electrode of each gradient film. Surface derivatization experiments were employed to investigate the concentration of residual ether units in the films. In addition, surface derivatization was used to investigate the reactivity of the gradient films toward primary amine groups in a graft copolymer of poly (L-lysine) and poly(ethylene glycol) (PLL-g-PEG copolymer) which was correlated to residual aldehyde, ketone and carboxylic acid functionalities within the films. The protein adsorption characteristics of the gradients were analyzed using three proteins of varying size and charge. Protein adsorption varied and was dependent on the chemistry and the physical properties (such as size and charge) of the proteins. A correlation between the concentration of ether functionality and the protein fouling characteristics along the gradient films was observed. The gradient coating technique developed in this work allows for the efficient and high-throughput study of biomaterial gradient coating interactions.

Improved biomolecule microarrays by printing on nanoporous aluminum oxide using a continuous-flow microspotter.

Biomolecules, including protein A, albumin, and immunoglobulin G, are spotted on top of a nanoporous substrate by using a continuous-flow microspotter (CFM) system, which normally produces spots 3 to 4 orders of magnitude more sensitive than conventional biomolecule printing methods. The spots are observed with a fluorescence scanner. By using the CFM to print spots on nanoporous substrates, an additional order of magnitude increase in signal is observed, which leads to high signal-to-background ratios, highly saturated spots, and a measurable signal at printing concentrations as low as 1.6 ng mL(-1). This technique produces highly concentrated biomolecular spots from dilute samples and significantly increases the sensitivity of sensing platforms.