RESUMO
Production of therapeutic monoclonal antibodies (mAbs) is a complex process that requires extensive analytical and bioanalytical characterization to ensure high and consistent product quality. Aggregation of mAbs is common and very problematic and can result in products with altered pharmacodynamics and pharmacokinetics and potentially increased immunogenicity. Rapid detection of aggregates, however, remains very challenging using existing analytical techniques. Here, we show a real-time and label-free fiber optical nanoplasmonic biosensor system for specific detection and quantification of immunoglobulin G (IgG) aggregates exploiting Protein A-mediated avidity effects. Compared to monomers, IgG aggregates were found to have substantially higher apparent affinity when binding to Protein A-functionalized sensor chips in a specific pH range (pH 3.8-4.0). Under these conditions, aggregates and monomers showed significantly different binding and dissociation kinetics. Reliable and rapid aggregate quantification was demonstrated with a limit of detection (LOD) and limit of quantification (LOQ) of about 9 and 30 µg/mL, respectively. Using neural network-based curve fitting, it was further possible to simultaneously quantify monomers and aggregates for aggregate concentrations lower than 30 µg/mL. Our work demonstrates a unique avidity-based biosensor approach for fast aggregate analysis that can be used for rapid at-line quality control, including lot/batch release testing. This technology can also likely be further optimized for real-time in-line monitoring of product titers and quality, facilitating process intensification and automation.
Assuntos
Antineoplásicos Imunológicos , Técnicas Biossensoriais , Imunoglobulina G/química , Anticorpos Monoclonais/química , Limite de Detecção , Técnicas Biossensoriais/métodos , Agregados ProteicosRESUMO
High sensitivity and cross-selectivity are mandatory properties for sensor arrays. Although metalloporphyrins and pH indicators are among the most common and appropriate choices for the preparation of optical sensor arrays, the sensitivity spectrum of these dyes is limited to those analytes able to induce an optical response. To extend the receptive field of optical sensors, we explore the design of composite materials, where the molecular interaction among the subunits enriches their sensing working mechanisms. We demonstrate that blends of single metalloporphyrins and pH indicators, tested with a transduction apparatus based on ubiquitous and easily available hardware, can be endowed with sensing properties wider than those of single constituents, enabling the recognition of a broad range of volatiles.
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Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Metaloporfirinas/química , Técnicas de Química Analítica , Processamento Eletrônico de Dados , Concentração de Íons de Hidrogênio , Processamento de Imagem Assistida por Computador , Fenômenos Ópticos , Sensibilidade e EspecificidadeRESUMO
Therapeutic monoclonal antibodies (mAbs) are critical for treatment of a wide range of diseases. Immunoglobulin G (IgG) is the most predominant form of mAb but is prone to aggregation during production. Detection and removal of IgG aggregates are time-consuming and laborious. Chromatography is central for purification of biopharmaceuticals in general and essential in the production of mAbs. Protein purification systems are usually equipped with detectors for monitoring pH, UV absorbance, and conductivity, to facilitate optimization and control of the purification process. However, specific in-line detection of the target products and contaminating species, such as aggregates, is currently not possible using convectional techniques. Here we show a novel fiber optical in-line sensor, based on localized surface plasmon resonance (LSPR), for specific detection of IgG and IgG aggregates during affinity chromatography. A flow cell with a Protein A sensor chip was connected to the outlet of the affinity column connected to three different chromatography systems operating at lab scale to pilot scale. Samples containing various IgG concentrations and aggregate contents were analyzed in-line during purification on a Protein A column using both pH gradient and isocratic elution. Because of avidity effects, IgG aggregates showed slower dissociation kinetics than monomers after binding to the sensor chips. Possibilities to detect aggregate concentrations below 1 % and difference in aggregate content smaller than 0.3 % between samples were demonstrated. In-line detection of aggregates can circumvent time-consuming off-line analysis and facilitate automation and process intensification.
Assuntos
Anticorpos Monoclonais , Cromatografia de Afinidade , Imunoglobulina G , Proteína Estafilocócica A , Ressonância de Plasmônio de Superfície , Imunoglobulina G/isolamento & purificação , Cromatografia de Afinidade/métodos , Cromatografia de Afinidade/instrumentação , Ressonância de Plasmônio de Superfície/métodos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Proteína Estafilocócica A/química , Agregados Proteicos , Concentração de Íons de HidrogênioRESUMO
Despite several types of fluorescent sensing molecules have been proposed and examined to signal Hg(2+) ion binding, the development of fluorescence-based devices for in-field Hg(2+) detection and screening in environmental and industrial samples is still a challenging task. Herein, we report the synthesis and characterization of three new coumarin-based fluorescent chemosensors featuring mixed thia/aza macrocyclic framework as receptors units, that is, ligands L1-L3. These probes revealed an OFF-ON selective response to the presence of Hg(2+) ions in MeCN/H2 O 4:1 (v/v), which allowed imaging of this metal ion in Cos-7 cells in vitro. Once included in silica core-polyethylene glycol (PEG) shell nanoparticles or supported on polyvinyl chloride (PVC)-based polymeric membranes, ligands L1-L3 can also selectively sense Hg(2+) ions in pure water. In particular we have developed an optical sensing array tacking advantage of the fluorescent properties of ligand L3 and based on the computer screen photo assisted technique (CSPT). In the device ligand L3 is dispersed into PVC membranes and it quantitatively responds to Hg(2+) ions in natural water samples.
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Cumarínicos/química , Corantes Fluorescentes/química , Mercúrio/análise , Nanopartículas/química , Espectrometria de Fluorescência , Animais , Células COS , Chlorocebus aethiops , Íons/química , Microscopia Confocal , Polietilenoglicóis/química , Cloreto de Polivinila/química , Teoria Quântica , Água/químicaRESUMO
A novel strategy for site-specific and covalent attachment of proteins has been developed, intended for robust and controllable immobilization of histidine (His)-tagged ligands in protein microarrays. The method is termed chelation assisted photoimmobilization (CAP) and was demonstrated using human IgG-Fc modified with C-terminal hexahistidines (His-IgGFc) as the ligand and protein A as the analyte. Alkanethiols terminated with either nitrilotriacetic acid (NTA), benzophenone (BP), or oligo(ethylene glycol) were synthesized and mixed self-assembled monolayers (SAMs) were prepared on gold and thoroughly characterized by infrared reflection absorption spectroscopy (IRAS), ellipsometry, and contact angle goniometry. In the process of CAP, NTA chelates Ni(2+) and the complex coordinates the His-tagged ligand in an oriented assembly. The ligand is then photoimmobilized via BP, which forms covalent bonds upon UV light activation. In the development of affinity biosensors and protein microarrays, site-specific attachment of ligands in a fashion where analyte binding sites are available is often preferred to random coupling. Analyte binding performance of ligands immobilized either by CAP or by standard amine coupling was characterized by surface plasmon resonance in combination with IRAS. The relative analyte response with randomly coupled ligand was 2.5 times higher than when site-specific attachment was used. This is a reminder that also when immobilizing ligands via residues far from the binding site, there are many other factors influencing availability and activity. Still, CAP provides a valuable expansion of protein immobilization techniques since it offers attractive microarraying possibilities amenable to applications within proteomics.
Assuntos
Quelantes/química , Histidina/química , Fragmentos Fc das Imunoglobulinas/química , Análise Serial de Proteínas , Histidina/análogos & derivados , Humanos , Ligantes , Estrutura Molecular , Processos FotoquímicosRESUMO
5,10,15,20-Tetraferrocenyl porphyrin, H2TFcP, a simple example of a donor-acceptor system, was tested as ligand for the development of a novel multi-transduction chemical sensors aimed at the determination of transition metal ions. The fluorescence energy transfer between ferrocene donor and porphyrin acceptor sub-units was considered. The simultaneously measured optical and potentiometric responses of solvent polymeric membranes based on H2TFcP permitted the detection of lead ions in sample solutions, in the concentration range from 2.7 × 10(-7) to 3.0 × 10(-3) M. The detection limit of lead determination was 0.27 µM, low enough to perform the direct analysis of Pb2+ in natural waters.
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Natural olfaction suggests that numerous replicas of small sensors can achieve large sensitivity. This concept of sensor redundancy can be exploited by use of optical chemical sensors whose use of image sensors enables the simultaneous measurement of several spatially distributed indicators. Digital image sensors split the framed scene into hundreds of thousands of pixels each corresponding to a portion of the sensing layer. The signal from each pixel can be regarded as an independent sensor, which leads to a highly redundant sensor array. Such redundancy can eventually be exploited to increase the signal-to-noise ratio. In this paper we report an algorithm for reduction of the noise of pixel signals. For this purpose, the algorithm processes the output of groups of pixels whose signals share the same time behavior, as is the case for signals related to the same indicator. To define these groups of pixels, unsupervised clustering, based on classification of the indicator colors, is proposed here. This approach to signal processing is tested in experiments on the chemical sensitivity of replicas of eight indicators spotted on to a plastic substrate. Results show that the groups of pixels can be defined independently of the geometrical arrangement of the sensing spots, and substantial improvement of the signal-to-noise ratio is obtained, enabling the detection of volatile compounds at any location on the distributed sensing layer.
Assuntos
Técnicas de Química Analítica , Compressão de Dados , Indicadores e Reagentes/análise , Compostos Orgânicos Voláteis/análise , AlgoritmosRESUMO
Therapeutic monoclonal antibodies (mAbs) provide new means for treatments of a wide range of diseases and comprise a large fraction of all new approved drugs. Production of mAbs is expensive compared to conventional drug production, primarily due to the complex processes involved. The affinity purification step is dominating the cost of goods in mAb manufacturing. Process intensification and automation could reduce costs, but the lack of real-time process analytical technologies (PAT) complicates this development. We show a specific and robust fiber optical localized surface plasmon resonance (LSPR) sensor technology that is optimized for in-line product detection in the effluent in affinity capture steps. The sensor system comprises a flow cell and a replaceable sensor chip functionalized with biorecognition elements for specific analyte detection. The high selectivity of the sensor enable detection of mAbs in complex sample matrices at concentrations below 2.5 µg mL-1. In place regeneration of the sensor chips allowed for continuous monitoring of multiple consecutive chromatographic separation cycles. Excellent performance was obtained at different purification scales with flow rates up to 200 mL min-1. This sensor technology facilitates efficient column loading, optimization, and control of chromatography systems, which can pave the way for continuous operation and automation of protein purification steps.
Assuntos
Anticorpos Monoclonais , Técnicas Biossensoriais , Cromatografia de Afinidade/métodos , Anticorpos Monoclonais/química , Ressonância de Plasmônio de SuperfícieRESUMO
Amyloid fibrils are composed of self assembled stacked peptide or protein molecules folded and trapped in a stable cross-beta-sheet conformation. The amyloid fibrillation mechanism represents an intriguing self-catalyzed process rendering replication of a molecular conformational memory of interest for prebiotic chemistry. Herein we describe how a solid surface can be rendered auto-catalytic for fibrillation of a protein solution. We have discovered that a hydrophobic silicon or glass surface can be made to continuously fibrillate solutions of insulin monomers under stressed conditions (pH 1.6, 65°C). It was found that the surface acts as a platform for the formation of nascent seeds that induce fibril replication on and at the surface. This autocatalytic effect stems from a layer a few insulin molecules thick representing an oligomeric layer of misfolded, conformationally trapped, insulin molecules that rapidly through epitaxial growth catalyze the rate determining step (nucleation) during fibril replication. This autocatalytic layer is generated by the protein-solid surface interaction and conformational changes of the adsorbed protein during exposure at the air-water interface. The resulting autocatalytic surface thus both initiates local conformational molecular self-replication and acts as a reservoir for fibril seeds budding off into solution spreading fibril replication entities to the surrounding medium. The possibility of catalysis of the conformational replication process by minute amounts of nucleation sites located on a recruiting surface can evade the issue of dramatic concentration dependence of amyloidogenesis.
Assuntos
Amiloide/química , Insulina/química , Amiloide/ultraestrutura , Animais , Catálise , Bovinos , Fluorescência , Vidro/química , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Transmissão , Microscopia de Polarização , Conformação Proteica , Silício/química , Soluções , Coloração e Rotulagem , Propriedades de SuperfícieRESUMO
Physiological investigations suggest that the olfactory mucosa probably plays an ancillary role in the recognition of odours introducing a sort of chromatographic separation that, together with the zonal distribution of olfactory receptors, gives place to selective spatio-temporal response patterns. It has been recently suggested that this behaviour may be simulated by chemical sensors embedded in continuous polymer layers. In this paper, in analogy to the biology of olfaction, a simple and compact platform able to separate and detect gases and vapours on the basis of their diffusion properties is proposed. In such a system, broadly selective colour indicators, such as metalloporphyrins, are embedded in continuous layers of polymers with different sorption properties. The exposure to various alcohols and amines shows that the porphyrins are mainly responsible for the recognition of the molecular family, while the occurring spatio-temporal signal patterns make possible the identification of the individual chemical species.
Assuntos
Cromatografia Gasosa/métodos , Polímeros/química , Corantes/química , Metaloporfirinas/química , Odorantes/análise , Mucosa Olfatória/fisiologiaRESUMO
Interest in the use of the optical properties of chemical indicators is growing steadily. Among the optical methods that can be used to capture changes in sensing layers, those producing images of large-area devices are particularly interesting for chemical sensor array development. Until now, few studies addressed the characterization of image sensors from the point of view of their chemical sensor application. In this paper, a method to evaluate such performance is proposed. It is based on the simultaneous measurement of absorption events in a metalloporphyrin layer with an image sensor and a quartz microbalance (QMB). Exploiting the well-known behaviour of QMB, comparison of signals enables estimation of the minimum amount of absorbed molecules that the image sensor can detect. Results indicate that at the single pixel level a standard image sensor (for example a webcam) can easily detect femtomoles of absorbed molecules. It should therefore be possible to design sensor arrays in which the pixels of images of large-area sensing layers are regarded as individual chemical sensors providing a ready and simple method for large sensor array development.
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We have with enthusiasm read the article "Calmangafodipir Reduces Sensory Alterations and Prevents Intraepidermal Nerve Fibers Loss in a Mouse Model of Oxaliplatin Induced Peripheral Neurotoxicity"[...].
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Nanostructured molecular assemblies may provide additional sensing properties not found in other arrangements of the same basic constituents. Among three-dimensional structures, nanotubes are particularly appealing for applications as chemical sensors, because of the potential inclusion of different guests inside the cavity or the induced modification of the skeletal interaction after analyte binding. Porphyrins are a class of compounds characterized by brilliant sensing properties, appearing also in non-ordered solid-state aggregates. In recent years, it was reported that aggregation of oppositely charged porphyrins led to the formation of self-assembled nanotubes and in this paper their sensing properties, both in solution and in the solid state, have been investigated. The interactions of porphyrin nanotubes with guest molecules have been monitored by following the changes in their UV-vis spectra. The results obtained have been exploited to build up a sensing platform based on a computer screen as a light source and a digital camera as detector. Porphyrin nanostructures exhibited an enhanced sensitivity to different compounds with respect to those shown by single porphyrin subunits. The reason for the increased sensitivity may be likely found in an additional sensing mechanism related to the modulation of the strength of the forces that keep the supramolecular ensemble together.
Assuntos
Cristalização/métodos , Gases/análise , Gases/química , Nanoestruturas/química , Nanotecnologia/métodos , Porfirinas/química , Espectrofotometria Ultravioleta/métodos , Substâncias Macromoleculares/química , Teste de Materiais , Conformação Molecular , Nanoestruturas/ultraestrutura , Tamanho da Partícula , Sensibilidade e Especificidade , Propriedades de SuperfícieRESUMO
Oxaliplatin typically causes acute neuropathic problems, which may, in a dose-dependent manner, develop into a chronic form of chemotherapy-induced peripheral neuropathy (CIPN), which is associated with retention of Pt2+ in the dorsal root ganglion. A clinical study by Coriat and co-workers suggests that co-treatment with mangafodipir [Manganese(II) DiPyridoxyl DiPhosphate; MnDPDP] cures ongoing CIPN. These authors anticipated that it is the manganese superoxide dismutase mimetic activity of MnDPDP that explains its curative activity. However, this is questionable from a pharmacokinetic perspective. Another, but until recently undisclosed possibility is that Pt2+ outcompetes Mn2+/Ca2+/Zn2+ for binding to DPDP or its dephosphorylated metabolite PLED (diPyridoxyL EthylDiamine) and transforms toxic Pt2+ into a non-toxic complex, which can be readily excreted from the body. We have used electron paramagnetic resonance guided competition experiments between MnDPDP (10logKML ≈ 15) and K2PtCl4, and between MnDPDP and ZnCl2 (10logKML ≈ 19), respectively, in order to obtain an estimate the 10logKML of PtDPDP. Optical absorption spectroscopy revealed a unique absorption line at 255 nm for PtDPDP. The experimental data suggest that PtDPDP has a higher formation constant than that of ZnDPDP, i.e., higher than 19. The present results suggest that DPDP/PLED has a high enough affinity for Pt2+ acting as an efficacious drug in chronic Pt2+-associated CIPN.
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Xenopus laevis melanophores can be used in high-throughput screens for guanine nucleotide binding protein coupled receptor ligands and have potential as biosensors. Inherent in this immortal cell line is a substantial variability, which macroscopic evaluations disregard. Here we demonstrate a systematic way to incorporate this natural variability in the evaluations. Clusters of similar cells from a sparsely seeded cell culture are examined by imaging changes in cell appearance, pigment motility, and cumulative displacements. The time evolution of the image intensity distributions of clusters upon a pigment-dispersing stimulus is used as a signature of the cell clusters, and their behaviors are classified by multivariate analysis. Conventional image subtraction procedures are used to highlight cumulative and transitory changes in the pigment dynamics, enabling characterization of multiple aspects of the cell response from a single experiment. Additionally, a simple way to accomplish standard optical density changes at the single-cell group level is shown. The present results also provide evidence that natural cell variability arising from a cell culture can enrich the diversity of responses from pigment-containing cells assays and underscore that in conventional macroscopic evaluations these aspects are overlooked and can lead to spurious results.
Assuntos
Melanóforos/fisiologia , Animais , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Fibroblastos/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Processamento de Imagem Assistida por Computador , Espaço Intracelular/ultraestrutura , Melanóforos/ultraestrutura , Xenopus laevisRESUMO
Melanophores from Xenopus laevis are pigmented cells, capable of quick colour changes through cyclic adenosine 3':5'-monophosphate (cAMP) coordinated transport of their intracellular pigment granules, melanosomes. In this study we use the melanophore cell line to evaluate the effects of Panax ginseng extract G115 on organelle transport. Absorbance readings of melanophore-coated microplates, Correlate-EIA direct cAMP enzyme immunoassay kit, and western blot were used to measure the melanosome movement and changes in intracellular signalling. We show that Panax ginseng induces a fast concentration-dependent anterograde transport of the melanosomes. No significant increase in the cAMP level was seen and pre-incubation of melanophores with the protein kinase C (PKC) inhibitor EGF-R Fragment 651-658 (M-EGF) only partly decreased the ginseng-induced dispersion. We also demonstrate that Panax ginseng, endothelin-3 (ET-3) and alpha-melanocyte stimulating hormone (MSH) stimulate an activation of mitogen activated protein kinase (MAPK). Pre-incubation with M-EGF decreased the MAPK activity induced by ET-3 and MSH, but again only marginally affected the response of Panax ginseng. Thus, in melanophores we suggest that Panax ginseng stimulates an anterograde transport of pigment organelles via a non-cAMP and mainly PKC-independent pathway.
Assuntos
Organelas/efeitos dos fármacos , Panax/química , Pigmentos Biológicos/metabolismo , Extratos Vegetais/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Western Blotting , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Endotelina-3/farmacologia , Técnicas Imunoenzimáticas/métodos , Melanóforos/efeitos dos fármacos , Melanóforos/metabolismo , Organelas/metabolismo , Extratos Vegetais/administração & dosagem , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Xenopus laevis , alfa-MSH/farmacologiaRESUMO
Dehydroabietic acid (DHAA) is a naturally occurring component of pine resin that was recently shown to open voltage-gated potassium (KV) channels. The hydrophobic part of DHAA anchors the compound near the channel's positively charged voltage sensor in a pocket between the channel and the lipid membrane. The negatively charged carboxyl group exerts an electrostatic effect on the channel's voltage sensor, leading to the channel opening. In this study, we show that the channel-opening effect increases as the length of the carboxyl-group stalk is extended until a critical length of three atoms is reached. Longer stalks render the compounds noneffective. This critical distance is consistent with a simple electrostatic model in which the charge location depends on the stalk length. By combining an effective anchor with the optimal stalk length, we create a compound that opens the human KV7.2/7.3 (M type) potassium channel at a concentration of 1 µM. These results suggest that a stalk between the anchor and the effector group is a powerful way of increasing the potency of a channel-opening drug.
Assuntos
Abietanos/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Canais de Potássio KCNQ/química , Superfamília Shaker de Canais de Potássio/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Humanos , Canais de Potássio KCNQ/metabolismo , Ligação Proteica , Superfamília Shaker de Canais de Potássio/metabolismo , Eletricidade Estática , XenopusRESUMO
A surface plasmon resonance (SPR) based flow chamber device was designed for real time detection of blood coagulation and platelet adhesion in platelet rich plasma (PRP) and whole blood. The system allowed the detection of surface interactions throughout the 6mm length of the flow chamber. After deposition of thromboplastin onto a section of the sensor surface near the inlet of the flow chamber, coagulation was detected downstream of this position corresponding to a SPR signal of 7 to 8 mRIU (7 to 8 ng/mm2). A nonmodified control surface induced coagulation 3.5 times slower. Platelet adhesion to gold and fibrinogen coated surfaces in the magnitude of 1.25 and 1.66 mRIU was also shown with platelets in buffer, respectively. SPR responses obtained with PRP and whole blood on surfaces that were methylated or coated with von Willebrand factor (vWF), fibrinogen, or collagen, coincided well with platelet adhesion as observed with fluorescence microscopy in parallel experiments. The present SPR detection equipped flow chamber system is a promising tool for studies on coagulation events and blood cell adhesion under physiological flow conditions, and allows monitoring of short-range surface processes in whole blood.
Assuntos
Técnicas Biossensoriais/instrumentação , Coagulação Sanguínea/fisiologia , Vasos Sanguíneos/fisiologia , Técnicas de Cultura de Células/instrumentação , Análise de Injeção de Fluxo/instrumentação , Adesividade Plaquetária/fisiologia , Ressonância de Plasmônio de Superfície/instrumentação , Técnicas Biossensoriais/métodos , Vasos Sanguíneos/citologia , Técnicas de Cultura de Células/métodos , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Análise de Injeção de Fluxo/métodos , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Resistência ao Cisalhamento , Ressonância de Plasmônio de Superfície/métodosRESUMO
Polyelectrolyte-coated nanoparticles consisting of terbium and gadolinium complexes with calix[4]arene tetra-diketone ligand were first synthesized. The antenna effect of the ligand on Tb(III) green luminescence and the presence of water molecules in the coordination sphere of Gd(III) bring strong luminescent and magnetic performance to the core-shell nanoparticles. The size and the core-shell morphology of the colloids were studied using transmission electron microscopy and dynamic light scattering. The correlation between photophysical and magnetic properties of the nanoparticles and their core composition was highlighted. The core composition was optimized for the longitudinal relaxivity to be greater than that of the commercial magnetic resonance imaging (MRI) contrast agents together with high level of Tb(III)-centered luminescence. The tuning of both magnetic and luminescent output of nanoparticles is obtained via the simple variation of lanthanide chelates concentrations in the initial synthetic solution. The exposure of the pheochromocytoma 12 (PC 12) tumor cells and periphery human blood lymphocytes to nanoparticles results in negligible effect on cell viability, decreased platelet aggregation and bright coloring, indicating the nanoparticles as promising candidates for dual magneto-fluorescent bioimaging.
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Distinctive generation of biochemical response patterns of eight different substances, using an assay based on pigment containing cells, was demonstrated. Xenopus laevis melanophores, transfected with human beta(2)-adrenergic receptor, were seeded in a 96 well microplate and used to generate individual biochemical images through a two transient measuring protocol that contributes to highlight the response signatures of the agents. Adequate signal processing creates distinctive patterns in a time-concentration response space suitable for substance classification. The concept of biochemical images is introduced here. The assays were evaluated both with a standard microplate reader and with a computer screen photo-assisted technique (CSPT) yielding similar results. Since CSPT platforms only demand standard computer sets and web cameras as measuring setup, applications for these kind of assays outside main-laboratories were discussed.