Gold Nanoparticles Used as Protein Scavengers Enhance Surface Plasmon Resonance Signal

Although several researchers had reported on methodologies for surface plasmon resonance (SPR) signal amplification based on the use of nanoparticles (NPs), the majority addressed the sandwich technique and low protein concentration. In this work, a different approach for SPR signal enhancement based on the use of gold NPs was evaluated. The method was used in the detection of two lectins, peanut agglutinin (PNA) and concanavalin A (ConA). Gold NPs were functionalized with antibodies anti-PNA and anti-ConA, and these NPs were used as protein scavengers in a solution. After being incubated with solutions of PNA or ConA, the gold NPs coupled with the collected lectins were injected on the sensor containing the immobilized antibodies. The signal amplification provided by this method was compared to the signal amplification provided by the direct coupling of PNA and ConA to gold NPs. Furthermore, both methods, direct coupling and gold NPs as protein scavengers, were compared to the direct detection of PNA and ConA in solution. Compared to the analysis of free protein, the direct coupling of PNA and ConA to gold NPs resulted in a signal amplification of 10-40-fold and a 13-fold decrease of the limit of detection (LOD), whereas the use of gold NPs as protein scavengers resulted in an SPR signal 40-50-times higher and an LOD 64-times lower.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2011/23895-8, 2017/01697-6]Univ Fed Sao Paulo, Inst Ciencia & Tecnol, Rua Talim 330, BR-12231280 Sao Jose Dos Campos, SP, BrazilUniv Fed Sao Paulo, Inst Ciencia & Tecnol, Rua Talim 330, BR-12231280 Sao Jose Dos Campos, SP, BrazilFAPESP [2011/23895-8, 2017/01697-6]Web of Scienc

Desenvolvimento de biossensor para deteção de biomarcadores de câncer de ovário baseado na técnica de SPRi

Considerado o câncer ginecológico mais letal, o câncer de ovário epitelial (COE) é em geral, diagnosticado na fase avançada resultando em uma sobrevida de 5 anos em 40 % dos casos. A falta de técnicas confiáveis que sejam capazes de detectar precocemente a doença desperta o interesse na busca de uma solução para este problema. Infelizmente, não há biomarcador específico e comumente é avaliado o nível sérico do CA-125 para diagnóstico e prognóstico da neoplasia. Embora este também não seja específico para COE, pode ser utilizado juntamente com a mesotelina aumentando a especificidade, visto que, estudos apontam que a interação dessas moléculas tem relação com o processo metastático da doença. Portanto, torna-se interessante o desenvolvimento de um biossensor capaz de detectar simultaneamente a mesotelina e o CA-125. O foco desta tese foi o desenvolvimento de um biossensor capaz de detectar simultaneamente e em baixas concentrações, as moléculas, CA -125 e mesotelina, tanto livre quanto associados entre si. O biossensor está baseado na técnica de Ressonância Plasmônica de Superfície (SPR) e sua alta sensibilidade foi alcançada pelo uso de nanopartículas de ouro (AuNPs) capazes de captar os biomarcadores nas amostras líquidas aumentando a concentração local dos analitos sobre o sensor e aumentando o sinal de SPR por acoplamento eletrônico. Uma vez desenvolvido o biossensor, esse poderá ser aplicado no estudo do possível uso de mesotelina e CA-125 como biomarcadores de COE no estádio inicial. O estudo analisou sucessivas injeções dos biomarcadores CA-125 e mesotelina, ambos em solução (intervalo analítico 9 nM a 120 nM). A partir desta análise foi obtido os respectivos limites de detecção ou quantificação: 3,03 nM e 13,62 nM. O biossensor detectou a mesotelina em uma concentração próxima do ponto de corte para COE (3 nM) e foi capaz de detectar o biomarcador CA-125 em baixa concentração (9 nM). Além disso, o biossensor interagiu preferencialmente e simultaneamente com os biomarcadores CA-125 e mesotelina incubados em soro fetal bovino. Por fim, o uso de AuNPs aumentou o sinal do sensor em 1,5 vezes para detecção de CA-125 se comparado a detecção direta e apresentou maior seletividade para ambos os biomarcadores. Portanto, o biossensor apresenta importantes características que viabiliza o teste com amostras reais e, em um futuro projeto, poderá ser usado no diagnóstico e prognóstico do COE.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fapesp 2016/0734

Aumento De Sensibilidade De Sensor De Ressonância Plasmônica De Superfície Para Detecção De Proteínas

This study describes the development of a method to increase the sensitivity of a Surface Plasmon Resonance Biosensor (SPR) to detect proteins at low concentrations. The method used gold nanoparticles (AuNPs) in two different approaches. In the first one, the protein to be detected was covalently linked to the AuNPs and then analyzed on the SPR sensor. In the second approach AuNPs were functionalized with specific antibody to capture protein of interest free in solution. The chosen proteins were lectins Arachis hypogaea (PNA) and Canavalia ensiformis (conA), and the biomarker of ovarian cancer, mesothelin. The second approach was more efficient in amplifying the signal, providing a better resolution response and reduced detection limit. The sensor response was compared to the detection of free protein in solution and the detection of protein covalently linked to AuNPs. In this order, the limit of detection for the approach of using the AuNPs to capture proteins was 98% and 89% lower in PNA detection and 79% and 77% lower in conA detection. It is possible that the higher efficiency was due to the role of AuNPs in enhancing the concentration of protein at NPs surface, as well as in providing the plasmonic coupling between the AuNPs and the gold surface of the SPR sensor. This result motivated the use of capture AuNPs in the detection of the protein mesothelin, an important biomarker used in the diagnosis of ovarian cancer and mesothelioma. The test was performed based on the cutoff point used to diagnose ovarian cancer (415pM). The result showed a detection limit of 35pM, which was of great relevance, demonstrating the efficiency of detection of mesothelin at low concentrations, demonstrating the potential use of this method for future studies in the area of cancer diagnosis.Este estudo descreve o desenvolvimento de um método para aumentar a sensibilidade de um biossensor de Ressonância Plasmônica de Superfície (SPR) para detecção de proteínas em baixas concentrações. O método utilizou nanopartículas de ouro (AuNPs) em duas metodologias. Em um primeiro método, a proteína a ser detectada foi ligada covalentemente às AuNPs e em seguida as NPs funcionalizadas foram analisadas no sensor de SPR. O Segundo método utilizou as AuNPs funcionalizadas com anticorpo específico para captura de proteína de interesse livre em solução. As proteínas utilizadas foram lectinas Arachis hypogaea (PNA) e Canavalia ensiformis (conA), e biomarcador de câncer de ovário, a mesotelina. O segundo método foi mais eficiente na amplificação do sinal resultando em uma resposta com melhor resolução e redução do limite de detecção. A resposta do sensor foi comparada com a detecção de proteína livre em solução e com a detecção de proteína ligada covalentemente às AuNPs. Nesta ordem, o limite de detecção foi 98% e 89% menor na detecção de PNA e 79% e 77% menor na detecção de conA. É possível que o segundo método tenha sido mais eficiente devido ao uso AuNPs como elemento de amplificação e capturador de proteína livre em solução, bem como por levar ao acomplamento plasmônico entre as AuNPs e a superfície de ouro do sensor SPR. O resultado verificado motivou o uso de AuNPs capturadoras na detecção da proteína mesotelina, um importante biomarcador utilizado no diagnóstico de câncer de ovário e mesotelioma. O teste foi realizado com base no ponto de corte utilizado para diagnosticar câncer de ovário (415pM). O resultado apresentou um limite de detecção de 35pM, esse valor foi de grande relevância demonstrando a eficiência na detecção de proteína mesotelina em baixas concentrações demonstrando o potencial uso desse método para futuros estudos na área de diagnóstico de câncer.Dados abertos - Sucupira - Teses e dissertações (2017

Gold Nanoparticles Used as Protein Scavengers Enhance Surface Plasmon Resonance Signal

Although several researchers had reported on methodologies for surface plasmon resonance (SPR) signal amplification based on the use of nanoparticles (NPs), the majority addressed the sandwich technique and low protein concentration. In this work, a different approach for SPR signal enhancement based on the use of gold NPs was evaluated. The method was used in the detection of two lectins, peanut agglutinin (PNA) and concanavalin A (ConA). Gold NPs were functionalized with antibodies anti-PNA and anti-ConA, and these NPs were used as protein scavengers in a solution. After being incubated with solutions of PNA or ConA, the gold NPs coupled with the collected lectins were injected on the sensor containing the immobilized antibodies. The signal amplification provided by this method was compared to the signal amplification provided by the direct coupling of PNA and ConA to gold NPs. Furthermore, both methods, direct coupling and gold NPs as protein scavengers, were compared to the direct detection of PNA and ConA in solution. Compared to the analysis of free protein, the direct coupling of PNA and ConA to gold NPs resulted in a signal amplification of 10–40-fold and a 13-fold decrease of the limit of detection (LOD), whereas the use of gold NPs as protein scavengers resulted in an SPR signal 40–50-times higher and an LOD 64-times lower

Interaction between nanoparticles, membranes and proteins: A surface plasmon resonance study

International audienceRegardless of the promising use of nanoparticles (NPs) in biomedical applications, several toxic effects have increased the concerns about the safety of these nanomaterials. Although the pathways for NPs toxicity are diverse and dependent upon many parameters such as the nature of the nanoparticle and the biochemical environment, numerous studies have provided evidence that direct contact between NPs and biomolecules or cell membranes leads to cell inactivation or damage and may be a primary mechanism for cytotoxicity. In such a context, this work was focused on the development of a fast and accurate method to characterize the interaction between NPs, proteins and lipidic membranes by surface plasmon resonance imaging (SPRi) technique. The interaction of gold NPs with mimetic membranes was evaluated by monitoring the variation of reflectivity after several consecutive gold NPs injections on the lipidic membranes prepared on the SPRi biochip. The interaction on the membranes with varied lipidic composition was compared regarding the total surface concentration density of gold NPs adsorbed on them. Then, the interaction of gold and silver NPs with blood proteins was analyzed regarding their kinetic profile of the association/dissociation and dissociation constants (koff). The surface concentration density on membrane composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine and cholesterol (POPC/cholesterol) was 2.5 times higher than the value found after the injections of gold NPs on POPC only or with dimethyldioctadecylammonium (POPC/DDAB). Regarding the proteins, gold 26 NPs showed a preferential binding to fibrinogen resulting in a value of variation of reflectivity 8 27 times higher than the value found for the other proteins. Differently, silver NPs showed similar 28 interaction on all the tested proteins but with a variation of reflectivity on immunoglobulin G (IgG) 29 2 times higher than the value found for the other tested proteins

Interaction between Nanoparticles, Membranes and Proteins: A Surface Plasmon Resonance Study

Regardless of the promising use of nanoparticles (NPs) in biomedical applications, several toxic effects have increased the concerns about the safety of these nanomaterials. Although the pathways for NPs toxicity are diverse and dependent upon many parameters such as the nature of the nanoparticle and the biochemical environment, numerous studies have provided evidence that direct contact between NPs and biomolecules or cell membranes leads to cell inactivation or damage and may be a primary mechanism for cytotoxicity. In such a context, this work focused on developing a fast and accurate method to characterize the interaction between NPs, proteins and lipidic membranes by surface plasmon resonance imaging (SPRi) technique. The interaction of gold NPs with mimetic membranes was evaluated by monitoring the variation of reflectivity after several consecutive gold NPs injections on the lipidic membranes prepared on the SPRi biochip. The interaction on the membranes with varied lipidic composition was compared regarding the total surface concentration density of gold NPs adsorbed on them. Then, the interaction of gold and silver NPs with blood proteins was analyzed regarding their kinetic profile of the association/dissociation and dissociation constants (koff). The surface concentration density on the membrane composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine and cholesterol (POPC/cholesterol) was 2.5 times higher than the value found after the injections of gold NPs on POPC only or with dimethyldioctadecylammonium (POPC/DDAB). Regarding the proteins, gold NPs showed preferential binding to fibrinogen resulting in a value of the variation of reflectivity that was 8 times higher than the value found for the other proteins. Differently, silver NPs showed similar interaction on all the tested proteins but with a variation of reflectivity on immunoglobulin G (IgG) 2 times higher than the value found for the other tested proteins