Gold Biosensor Research Articles

A mesoporous gold biosensor to investigate immune checkpoint protein heterogeneity in single lung cancer cells

Immune checkpoint proteins (ICPs) play a major role in a patient's immune response against cancer. Tumour cells usually express those proteins to communicate with immune cells as a process of escaping the anti-cancer immune response. Detecting the major functional immune checkpoint proteins present on cancer cells (such as circulating tumor cells or CTCs) and examining the heterogeneity in their expression at the single-cell level could play a crucial role in both cancer diagnosis and the monitoring of therapy. In this study, we develop a mesoporous gold biosensor to precisely assess ICP heterogeneity in individual cancer cells within a lung cancer model. The platform utilizes a nanostructured mesoporous gold surface to capture CTCs and a Surface Enhanced Raman Scattering (SERS) readout to identify and monitor the expression of key ICP proteins (PD-L1, B7H4, CD276, CD80) in lung cancer cells. The homogeneous and abundant pores in mesoporous 3D gold nanostructures enable increased antibody loading on-chip and an enhanced SERS signal, which are key to our single cell capture, and accurate analysis of ICPs in cancer cells with high sensitivity. Our lung cancer cell line model data showed that our method can detect single cells and analyse the expression of four lung cancer associated ICPs on individual cell surfaces during treatment. To show the potential of our mesoporous gold biosensor in analysing clinical samples, we tested 9 longitudinal Peripheral Blood Mononuclear Cells (PBMC) samples from lung cancer patient before and after therapy. Our mesoporous biosensor successfully captured single CTCs and found that the expression of ICPs in CTCs is highly heterogeneous in both pre-treatment and treated PBMC samples isolated from lung cancer patient blood. We suggest that our findings will help clinicians in selecting the most appropriate therapy for patients.

Discriminating Glioblastoma from Peritumoral Tissue by a Nanohole Array-Based Optical and Label-Free Biosensor

In glioblastoma (GBM) patients, maximal safe resection remains a challenge today due to its invasiveness and diffuse parenchymal infiltration. In this context, plasmonic biosensors could potentially help to discriminate tumor tissue from peritumoral parenchyma based on differences in their optical properties. A nanostructured gold biosensor was used ex vivo to identify tumor tissue in a prospective series of 35 GBM patients who underwent surgical treatment. For each patient, two paired samples, tumor and peritumoral tissue, were extracted. Then, the imprint left by each sample on the surface of the biosensor was individually analyzed, obtaining the difference between their refractive indices. The tumor and non-tumor origins of each tissue were assessed by histopathological analysis. The refractive index (RI) values obtained by analyzing the imprint of the tissue were significantly lower (p = 0.0047) in the peritumoral samples (1.341, Interquartile Range (IQR) 1.339–1.349) compared with the tumor samples (1.350, IQR 1.344–1.363). The ROC (receiver operating characteristic) curve showed the capacity of the biosensor to discriminate between both tissues (area under the curve, 0.8779, p < 0.0001). The Youden index provided an optimal RI cut-off point of 0.003. The sensitivity and specificity of the biosensor were 81% and 80%, respectively. Overall, the plasmonic-based nanostructured biosensor is a label-free system with the potential to be used for real-time intraoperative discrimination between tumor and peritumoral tissue in patients with GBM.

The signal conversion strategy using the lytic transglycosylase Cag4-double nanoporous gold co-catalysis for the rapid screening of drugs against Helicobacter pylori infection

The cag pathogenicity island (cagPAI) is the main virulence factor of gastric carcinoma induced by Helicobacter pylori (H. pylori). The lytic transglycosylase Cag4 is an important component that assists in the translocation of the bacterial oncoprotein CagA and maintains the peptidoglycan cycle. The allosteric regulation of Cag4 has been preliminarily demonstrated to inhibit H. pylori infection. Unfortunately, a rapid screening technology for allosteric regulators of Cag4 has not been established. In this study, a novel Cag4-double nanoporous gold (NPG) biosensor based on enzyme-inorganic co-catalysis was constructed using the heterologously expressed H. pylori 26695 Cag4 as the biological recognition element for screening Cag4 allosteric regulators. The results showed that chitosan or carboxymethyl chitosan was a mixed Cag4 inhibitor combining non-competition with uncompetition. The inhibition constants were Ki' Chitosan = 0.88909 mg/mL and Ki' Carboxymethyl chitosan = 1.13480 mg/mL, respectively. Surprisingly, D-(+)-cellobiose showed the activation effect of Cag4 on E. coli MG1655 cell wall lysis by decreasing the Ka value by 29.7% and increasing the Vmax value by 71.3%. In addition, molecular docking revealed the importance of the polarity of the C2 substituent group with glucose as the main structure in the Cag4 allosteric regulator. This study provides a fast and useful platform for screening potential new drugs based on the Cag4 allosteric regulator.

Comparison of Gold Biosensor Combined with Light Microscope Imaging System with ELISA for Detecting Salmonella in Chicken after Exposure to Simulated Chilling Condition.

In this study, the performance of a gold biosensor combined with light microscope imaging system (GB-LMIS) was comparatively evaluated against enzyme-linked immunosorbent assay (ELISA) for detecting Salmonella under simulated chilling condition. The optimum concentration of anti-Salmonella polyclonal antibodies (pAbs) was determined to be 12.5 and 100 μg/ml for ELISA and GB-LMIS, respectively. GB-LMIS exhibited a sufficient and competitive specificity toward three tested Salmonella among only. To mimic a real-world situation, chicken was inoculated with Salmonella cocktail and stored under chilling condition for 48 h. The overall growth of Salmonella under chilling condition was significantly lower than that under non-exposure to the chilling condition (p < 0.05). No significant differences in bacterial growth were observed between brain heart infusion and brilliant green broth during the enrichment period (p > 0.05). Finally, both GB-LMIS and ELISA were employed to detect Salmonella at every 2-h interval. GB-LMIS detected Salmonella with a competitive specificity by the direct observation of bacteria on the sensor using a charge-coupled device camera within a detection time of ~2.5 h. GB-LMIS is a feasible, novel, and rapid method for detecting Salmonella in poultry facilities.

Study on Postoperative Nursing of Patients with Gastric Diseases Based on Nano Gold Biosensor.

During the interventional operation of patients with gastric disease under nano gold biosensor, the nursing staff should have professional nursing knowledge. High quality nursing is a kind of nursing intervention based on holistic nursing and patient-centered. It can make corresponding nursing measures according to the actual condition of patients, so that patients can feel high-quality nursing services. In this study, high-quality nursing measures were taken for patients with gastric diseases in the interventional treatment of nano gold biosensor. Before the operation, relevant psychological nursing measures were given to patients, and related knowledge of disease and operation were explained. The skillful movements in operation and the cordial sympathy after operation can appease the patients' bad psychological state to a certain extent, so as to effectively reduce the patients' anxiety, depression and other psychological emotions. High quality nursing intervention for patients with gastric diseases under the intervention of nano gold biosensor can effectively improve the degree of anxiety and depression of patients, the recovery of bowel sounds and the time of getting out of bed activities, improve the treatment compliance, and promote the early recovery of patients' health.

Nanostructured mesoporous gold biosensor for microRNA detection at attomolar level

Advances in nanoarchitectonics enable a wide variety of nanostructured electrodes with tunable shapes and surface for constructing sensitive biosensors. Herein we demonstrate the fabrication of a mesoporous gold (Au) biosensor for the specific and sensitive detection of miRNA in a relatively simple and portable manner. The electrocatalytic activity of the mesoporous Au electrode (MPGE) towards the redox reaction of Fe(CN)6]3-/4- expansively examined. Leveraging the electrocatalytic activity and signal enhancement capacity of the MPGE, an ultrasensitive and specific electrochemical sensor was developed for the detection of microRNA (miRNA). The target miRNA from spiked samples is selectively isolated and purified using magnetic bead-capture probe followed by the direct adsorption on the MPGE through direct affinity interaction between miRNA and mesoporous Au surface. The MPGE-bound miRNA is then quantified by differential pulse voltammetry (DPV) using [Fe(CN)6]4-/3- redox system (Faradaic current decrease with reference to the bare MPGE). This method evades the cumbersome PCR (polymerase chain reaction) and enzymatic amplification steps. This is a single-step assay building which can detect a wide dynamic linear range (100 aM to 1 nM) of miRNA with an ultra-low limit detection of 100 aM and present high translational potentiality for the development of high-performance detection tools for clinics.

Characterisation of the bacterial biosensor GMG in E‐coli BL21 (DE3)

In this work, a gold biosensor has been constructed and characterised in Escherichia coli (E. coli). Though gold biosensors have been previously reported, a NOT gate gold biosensing genetic circuit was constructed using an orthogonal inverter system as an alternative to a circuit where output is contingent solely on input. This enables the detection of changes in gold concentration whereby circuit remains OFF in presence of inducer and ON in absence of inducer. The proposed biosensor is an alternative to conventional forms of gold detection such as Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). The constructed biosensor is a collection of components – golTSB operon (isolated from Salmonella enterica), mf-Lon protease (isolated from Mesoplasma florum) and associated ssrA degradation tag with GFP acting as a reporter. The minimum time and concentration required by the biosensor gol-mflon-GFP (GMG) to achieve the ON output is characterised here using plate reader data derived from a single multi-stage assay.

Simultaneous assessment of cell morphology and adhesion using aluminum nanoslit-based plasmonic biosensing chips

A variety of physiological and pathological processes rely on cell adhesion, which is most often tracked by changes in cellular morphology. We previously reported a novel gold nanoslit-based biosensor that is capable of real-time and label-free monitoring of cell morphological changes and cell viability. However, the preparation of gold biosensors is inefficient, complicated and costly. Recently, nanostructure-based aluminum (Al) sensors have been introduced for biosensing applications. The Al-based sensor has a longer decay length and is capable of analyzing large-sized mass such as cells. Here, we developed two types of double-layer Al nanoslit-based plasmonic biosensors, which were nanofabricated and used to evaluate the correlation between metastatic potency and adhesion of lung cancer and melanoma cell lines. Cell adhesion was determined by Fano resonance signals that were induced by binding of the cells to the nanoslit. The peak and dip of the Fano resonance spectrum respectively reflected long- and short-range cellular changes, allowing us to simultaneously detect and distinguish between focal adhesion and cell spreading. Also, the Al nanoslit-based biosensor chips were used to evaluate the inhibitory effects of drugs on cancer cell spreading. We are the first to report the use of double layer Al nanoslit-based biosensors for detection of cell behavior, and such devices may become powerful tools for anti-metastasis drug screening in the future.

Advanced fabrication of biosensor on detection of Glypican-1 using S-Acetylmercaptosuccinic anhydride (SAMSA) modification of antibody

Glypican-1 (GPC-1) has been recognized as biomarker of pancreatic cancer. Quantification of GPC-1 level is also pivotal to breast cancer and prostate cancer’s patients. We hereby report the first biosensor for GPC-1 detection. Instead of using crosslinking technique and surface immobilization of antibody, we applied a novel method for biosensor fabrication, using S-Acetylmercaptosuccinic anhydride (SAMSA) to modify the Anti-GPC-1 producing a thiol-linked Anti-GPC-1. The thiol-linked Anti-GPC-1 was then directly formed a single-layer antibody layer on the gold biosensor, minimizing the biosensor preparation steps significantly. Time of Flight Secondary Ions Mass Spectroscopy (TOF-SIMS) characterization verified the thiol-linked antibody layer and demonstrated a unique perspective for surface protein characterization. Differential pulse voltammetry (DPV) was applied to quantify GPC-1 antigen in undiluted human serum with a concentration range of 5,000 pg/µL to 100 pg/µL. The performance of this newly designed biosensor was also compared with modified self-assembled monolayer system fabricated biosensor, demonstrating the high-sensitivity and high-reproducibility of the SAMSA modified antibody based biosensor. This simple fabrication method can also expand to detection of other biomolecules. The simplified operation process shows great potential in clinical application development.

Production and characterization of anti-Salmonella polyclonal antibodies as bio-recognition element for developing a microbial monitoring method

For the construction of the microbial monitoring method, anti-Salmonella polyclonal antibodies (pAbs) were produced from a rabbit and purified by saturated ammonium sulfate precipitation and protein A affinity column. The reactivity of anti-Salmonella pAbs was compared to that of commercial ones by using an indirect ELISA. The specificity of anti-Salmonella pAbs was investigated using 20 Salmonella serotypes and 20 non-Salmonella strains. A capturing ability of anti-Salmonella pAbs was investigated by exposing antibody-immobilized gold biosensor to different concentration of Salmonella mixture. Anti-Salmonella pAbs were successfully produced and purified with an antibody concentration of 2.0 mg/mL The reactivity of purified anti-Salmonella pAbs was greater than that of commercial one at all tested concentrations. All Salmonella serotypes, except S. Diarizonae, showed excellent binding efficiency with purified anti-Salmonella pAbs. Moreover, the purified anti-Salmonella pAbs showed excellent specificity against all non-Salmonella strains. The anti-Salmonella pAbs immobilized on the gold biosensor demonstrated the successful capturing capability against Salmonella with a dose-response manner. Therefore, the anti-Salmonella pAbs exhibited sufficient reactivity, specificity, as well as capturing capability against Salmonella to be considered as a bio-recognition element.

Determination of best enrichment media for growth of Salmonella injured from cold temperature during process and storage

This purpose of this study was to determine the best enrichment medium for rejuvenating and recovering Salmonella placed in cold temperature prior to the employment of the gold biosensor combined with a light microscopic imaging system. A mixture of nalidixic-resistant Salmonella Typhimurium and Enteritidis were inoculated onto chicken (1,000 CFU/chicken). After cold injury at 4℃ for 24 hr, Salmonella on chicken was enriched for 6 hr with six non-selective media including buffered peptone water broth, lactose broth, brain heart infusion broth (BHI), universal pre-enrichment broth, nutrient broth, and tryptic soy broth, and five selective media including brilliant green broth (BG), rappaport-vassiliadis R10 broth, selenite cystine broth, selenite broth, and tetrathionate brilliant green broth (TBG) for the comparison of Salmonella growth. Various concentrations of Salmonella (10, 50, 100, 500, and 1,000 CFU/chicken) were then enriched for 6 hr in both BHI and BG media to select the best media. BHI was selected as the most effective non-selective enrichment medium, while BG was selected as the most effective selective enrichment medium. Finally, BHI medium was selected as the most efficient enrichment medium for Salmonella growth injured from cold temperature during processing or storage.

Nanostructured gold dsDNA sensor for early detection of breast cancer by beta protein 1 (BP1)

Beta protein 1 (BP1) is a homeobox protein expressed in 80% of breast cancer cells in either estrogen receptor (ER) positive or ER negative breast cancer. However, it is barely detectable in normal breast tissues. In this project we present an electrochemical DNA nanostructured gold biosensor for detection of BP1. The gold sensor is first electrochemically nanostructured in 0.5M sulfuric acid to reach superior conductivity, larger surface area, and higher stability. Nanostructured gold surface was characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The nanostructured gold sensor is then modified with double-stranded (ds) DNA mapping the genomic sequence that contains the binding site for BP1. A redox-active probe (methylene blue) was intercalated in dsDNA to monitor the binding event of BP1. A linear correlation of the electrochemical response by concentration of BP1 was obtained (R2=0.998) with a limit of detection of 1.2nM. This nanostructured gold dsDNA sensor is shown to be sensitive, selective, stable, and reusable allowing for its potential clinical use.

Determination of binding capacity and adsorption enthalpy between Human Glutamate Receptor (GluR1) peptide fragments and kynurenic acid by surface plasmon resonance experiments

The interaction between kynurenic acid (KYNA) and two peptide fragments (ca. 30 residues) of Human Glutamate Receptor 201–300 (GluR1) using surface plasmon resonance (SPR) spectroscopy was investigated. Because of the medical interest in the neuroscience, GluR1 is one of the important subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR). AMPARs are ionotoropic glutamate receptors, which are mediating fast synaptic transmission and are crucial for plasticity in the brain. On the other hand, KYNA has been suggested to have neuroprotective activity and it has been considered for apply in therapy in certain neurobiological disorders. In this article the adsorption of the GluR1201–230 and GluR1231–259 peptides were studied on gold biosensor chip. The peptides were chemically bonded onto the gold surface via thiol group of l-cysteine resulted in the formation of peptide monolayer on the SPR chip surface. Because the GluR1231–259 peptide does not contain l-cysteine the Val256 was replaced by Cys256. The cross sectional area and the surface orientation of the studied peptides were determined by SPR and theoretical calculations (LOMETS) as well. The binding capability of KYNA on the peptide monolayer was studied in the concentration range of 0.1–5.0mM using 150mM NaCl ionic strength at pH 7.4 (±0.02) in phosphate buffer solutions. In order to determine the binding enthalpy the experiments were carried out between +10°C and +40°C. The heat of adsorption was calculated by using adsorption isotherms at different surface loading of KYNA on the SPR chip.

Correction: A Whole-Cell Biosensor for the Detection of Gold

The authors wish to acknowledge an error in and and correct an error in their use of a reference. The fourth sentence of the seventh paragraph of the Introduction should read: A biosensor system, observed by Checa et al. (2011), was capable of detecting Au, quantification of Au was not reported here: However, quantification is crucially important for geochemical exploration [26]. In addition, the authors wish to acknowledge the relevance of an article 'Cerminati S, Soncini FC, Checa SK. Selective detection of gold using genetically engineered bacterial reporters. Biotechnol Bioeng. 2011 Nov; 108(11):2553-60. doi: 10.1002/bit.23213', which relates to the results described in the present article, as follows: A Au biosensor system based on the golTSB regulon and a fluorescent reporter protein was previously developed by Cerminati et al. (2011) and dose-response curves were constructed using KAu(CN)2. While Cerminati et al. (2011) suggested that biosensor systems may be useful for quantifying Au in real soil samples this was not tested in their study. Cerminati et al. (2011) developed a Au biosensor system which under clean laboratory conditions was capable of detecting environmentally relevant levels of Au. Our study takes further steps towards the development of a field-ready biosensor system with the development and testing of a selective extraction technique for Au, thermodynamic modelling of Au complexes under experimental conditions and the electrochemical testing of the sensor on complex environmental samples. Similar to the biosensor system presented in Zammit et al. (2013), the Au biosensor developed by Cerminati et al. (2011) was able to quantify Au. However, the gold biosensor developed by Cerminati et al. (2011) itself and the experimental conditions in the Checa et al. (Checa SK, Espariz M, Perez Audero ME, Botta PE, Spinell SV, et al. (2007) Bacterial sensing of and resistance to gold salts. Mol. Mic. 63: 1307-1318.) and Cerminati et al. (2011) studies varied in a number of important aspects from the ones in Zammit et al. (2013). The Cerminati et al. (2011) biosensor was developed using the golTSB genes from S. typhimurium fused to the gene of a green fluorescent protein (GFP) and expressed in E. coli. In contrast, the biosensor developed for this study used the golTSB genes from S. typhimurium transcriptionally fused to a promoterless lacZ reporter cassette, whose activity was measured electrochemically aimed at future in-field-application. These differences may have contributed to the differences in sensitivity of Au detection reported in both studies. The biosensor developed by Cerminati et al. (2011) was able to quantify Au down to 33.23 nM Au(I). The biosensor developed by Zammit et al. (2013) was able to quantify Au down to a detection limit of 10 nM. However, differences may also be a result of the different Au(I/III)-complexes used in both studies to construct standard curves. While KAu(CN)2 was used by Cerminati et al. (2011), Au(I)-thiosulfate and AuHCl4.3H2O were added to the medium by Zammit et al. (2013). The concentrations of metals tested ranged from 10 *M of Hg(II) to 1 mM of Cu(II) (50 *M of Au was used). These concentrations were not normalized in subsequent analyses. Importantly, in Zammit et al. (2013), cross reactivity of Au(I/III) was tested by mixing the complexes mixed with the other metal ions to show that Au can still be detected by the sensor whereas the Checa et al. (2007) and Cerminati et al. (2011) studies only investigated one metal at a time.

Connaissances et pratiques en matière de dépistage du cancer du sein des femmes en Tunisie

We report an enzyme-modified nanoporous gold (NPG) biosensor with excellent sensitivity and selectivity towards the detection of glucose in the physiological environment containing common interferents. The internal surface of biocompatible NPG films is functionalized with glucose oxidase using thiols as linking molecules. The three-dimensional porous metal/organic hybrid electrode exhibits effective enzyme immobilization and peroxide detection on gold ligaments for amperometric glucose sensing. This study has important implications in developing NPG based metal/organic electrodes for electrochemical sensors with high selectivity and sensitivity.

Biofunctionalized nanoporous gold for electrochemical biosensors

We report an enzyme-modified nanoporous gold (NPG) biosensor with excellent sensitivity and selectivity towards the detection of glucose in the physiological environment containing common interferents. The internal surface of biocompatible NPG films is functionalized with glucose oxidase using thiols as linking molecules. The three-dimensional porous metal/organic hybrid electrode exhibits effective enzyme immobilization and peroxide detection on gold ligaments for amperometric glucose sensing. This study has important implications in developing NPG based metal/organic electrodes for electrochemical sensors with high selectivity and sensitivity.

Rapid Detection of E. coli O157:H7 on Turnip Greens Using a Modified Gold Biosensor Combined with Light Microscopic Imaging System

This research aims to demonstrate the feasibility of a modified gold biosensor to detect E. coli O157:H7 in leafy turnip greens. The gold biosensor was modified with dithiobis-succinimidyl propionate (DSP) and/or protein A or G. The gold biosensor modified with DSP (Gold-DSP) was combined with a light microscopic imaging system (LMIS). The optimal concentration and specificity of anti-E. coli O15 polyclonal antibodies (pAbs) on the biosensor were determined. The reliability of Gold-DSP biosensor was investigated by determining the sensitivity, specificity, and limit of detection (LOD) of the Gold-DSP combined with LMIS. The Gold-DSP combined with LMIS was applied to turnip greens for E. coli O157:H7 detection. The modification of Gold biosensor with DSP significantly increased the detected number of E. coli O157:H7. The specificity of pAbs was sufficient to react with target E. coli O157:H7 among the tested bacterial culture. The optimum concentration of pAbs was determined as 200 μg/mL. The sensitivity, specificity, and LOD of Gold-DSP combined with LMIS were determined as 100%, 90%, and 10(3) CFU/25 mm(2) , respectively. When applied to turnip greens, the Gold-DSP combined with LMIS could detect 2641 ± 394 and 15383 ± 3853 cell/mm(2) with the initial concentrations of 10(1) and 10(2) CFU/25 g turnip greens, respectively, after 10 h-enrichment. Overall, this research suggested that the Gold-DSP combined with LMIS could be used to detect E. coli O157:H7 on turnip greens qualitatively and quantitatively.

Green glycosylation using ionic liquid to prepare alkyl glycosides for studying carbohydrate–protein interactions by SPR

Several simple glycosides of D-glucose (Glc) and N-acetyl-D-galactosamine (GalNAc) were prepared in a single step glycosylation reaction using unprotected and unactivated sugar donors. The resulting GalNAc glycoside, containing a bifunctional linker, was used to immobilize this glycoconjugate to a self assembled monolayer on a gold biosensor chip. Surface plasmon resonance (SPR) experiments demonstrated that this immobilized glycoconjugate bound to GalNAc specific lectin, Viscum album agglutinin.

Two examples of nanostructured gold surfaces as biosensors. Surface-enhanced chemiluminescence and double detection by surface plasmon resonance and luminescence

In this paper, we present a review of our activities in the field of gold biosensors. Nanostructured gold surfaces can be used in biology for their plasmonic and/or catalytic properties. In a first part, we show how the gold plasmonic properties allow the detection of the biotin-streptavidin binding by two types of techniques: extinction spectroscopy and also luminescence spectroscopy when the probe (here the streptavidin) is labelled by polysiloxane particles encapsulating fluorophores. In a second part, we demonstrate that the catalytical properties of gold corrugated surfaces can significantly enhance the chemiluminescence of luminol brought at vicinity. We found that the surface-enhancement induced by gold is two times of magnitude greater than that induced by silver.

Novel biosensor chip for simultaneous detection of DNA-carcinogen adducts with low-temperature fluorescence

A monoclonal antibody (MAb)–gold biosensor chip with low-temperature laser-induced fluorescence detection for analysis of DNA-carcinogen adducts is described. Optimization of the detection limit, dynamic range, and biosensing applicability of the MAb–gold biosensor chip was achieved by: (1) using dithiobis(succinimidyl propionate (DSP)) as a protein linker and (2) employing recombinant protein A to provide oriented immobilization of the MAbs. The use of DSP, which has a short methylene chain length, led to faster protein binding kinetics and higher protein surface density than a longer dithiobis(succinimidyl undecanoate) (DSU) linker. The incorporation of recombinant protein A increased the distance between the oriented MAb-bound analytes and the gold surface. The increased distance minimized fluorescence quenching, resulting in about a 10-fold increase in the fluorescence signal in comparison with a chip without protein A. The improved chip architecture was used to demonstrate that biosensing of two structurally similar benzo[ a]pyrene (BP)-derived DNA adducts, BP-6-N7Gua and BP-diolepoxide-10-N 2dG, bound to two specific MAbs immobilized from a mixture at the same address on the chip, is feasible. These mutagenic adducts are formed by one-electron oxidation and monooxygenation pathways, and are depurinating and stable DNA adducts, respectively. It is shown that the DNA adducts can be easily identified at the same address using time-resolved, low-temperature laser-based fluorescence spectroscopy. The current limit of detection is in the low femtomole range. These results indicate that a single biosensor chip consisting of a Au/DSP/protein A/MAb nanoassembly, with analyte-specific MAbs and low-temperature fluorescence detection should be suitable for simultaneous detection and quantitation of the above adducts, as well as the luminescent antigens for which selective MAbs exist.