Disposable amperometric magnetoimmunosensors for the specific detection of Streptococcus pneumoniae
Introduction
Streptococcus pneumoniae (pneumococcus) is an important human pathogen that causes meningitis, otitis media, sepsis, and pneumonia. This pathogen, together with Haemophilus influenzae type b, is currently the leading cause of bacterial meningitis in children and adults (Editorial, 2009). Pneumonia kills more children under 5 years of age worldwide than any other infectious disease. This disease kills an estimated 2 million children in this age group and causes 14.5 million cases of serious illness each year, more than HIV/AIDS, measles, and malaria combined. In an era of increasing resistance to antimicrobial agents, accurate identification and rapid diagnosis of pneumococcal infections are critical and may have important implications in the selection of antimicrobial agents for therapy (Dowell et al., 2001).
The golden standard procedure for establishing an etiological diagnosis includes Gram stain and culture of blood, sputum or cerebrospinal fluid (CSF) samples. The Gram stain examination is rapid but scarcely sensitive (>105 bacteria mL−1) and culture takes 24–48 h and is sometimes compromised by spontaneous autolysis or antibiotic treatment (Stuertz et al., 1998, Rai et al., 2004, Rouphael et al., 2008). Alternatives to these methods are direct pneumococcal antigen detection in body fluids by latex agglutination, co-agglutination, counter immunoelectrophoresis, enzyme-linked immunosorbent assay (ELISA) (García-Suárez et al., 2007, García-Suárez et al., 2009), DNA probes, polymerase chain reaction methods (Corless et al., 2001, Stralin et al., 2006, Harris et al., 2008), and several pneumococcal antigen tests (Stuertz et al., 1998, Samra et al., 2003, Ehara et al., 2008, Rai et al., 2004). An immunochromatographic membrane assay, Binax NOW S. pneumoniae Urinary Antigen Test (Inverness Medical International, Cranfield, UK), detects the C polysaccharide cell wall antigen common to all S. pneumoniae isolates. This test is commercially available, rapid (≅15 min), easy-to-use and was approved by the US Food and Drug Administration (FDA) for use in the United States (Dowell et al., 2001, Samra et al., 2003). The efficiency of this technique does not depend on treatment with antibiotics (Andreo et al., 2006, Lasocki et al., 2006) and it has demonstrated to be accurate for direct urine and CSF testing. However, it has a low sensitivity (the manufacturer specifies a detection limit of about 105 cfu mL−1 in CSF (Gisselsson-Solén et al., 2007), a poor selectivity towards other streptococci (Streptococcus mitis, Streptococcus oralis and other α-hemolytic streptococci), and various pathogenic bacteria (Staphylococcus aureus and H. influenzae) including Escherichia coli (Navarro et al., 2004, Moore et al., 2004, Lasocki et al., 2006, Gisselsson-Solén et al., 2007). Furthermore, this test fails to distinguish infants and children (which are more likely to be asymptomatic carriers of pneumococci than adults) with pneumococcal pneumonia from those who are merely colonized (Dowell et al., 2001, Samra et al., 2003, Navarro et al., 2004, Andreo et al., 2006). Also, it is associated with a lack of detection immediately after the onset of infection, and with long-term antigen-positive results regardless of treatment (1–3 months) or if the patient has been vaccinated with a pneumococcal conjugate vaccine shortly before the test (Ehara et al., 2008). Given the foremost interest associated with detecting quickly and unequivocally the presence of low concentrations of S. pneumoniae in clinical samples and the limitations of the available methodologies, there is an urgent need for developing novel diagnostic approaches that may offer a more sensitive, specific, and rapid detection of S. pneumoniae.
An alternative approach is the use of electrochemical immunosensors which combine the high selectivity provided by immunological species with the high sensitivity achieved with modern electrochemical techniques (Zacco et al., 2006). Recently, improvements in immunosensor performance, including enhanced sensitivity and reduced detection time, were attributed to the use of magnetic beads (MBs) (Nam et al., 2003, Bange et al., 2005, Rosi and Mirkin, 2005, Marquette and Blum, 2006, Zhang et al., 2006, Willner et al., 2007). MBs allow easy separation and localization of target analytes by an external magnet, fast immunoreactions between antigen and antibody, and low nonspecific binding by surface modification (Richardson et al., 2001, Matsunaga and Okamura, 2002, Zacco et al., 2006, Hsing et al., 2007, Selvaraju et al., 2008, Barletta et al., 2009). These approaches separate the steps related to the immunoreaction from the one concerning the electrochemical detection, which facilitates the transduction of the affinity event (Centi et al., 2007).
This paper describes, for the fist time, the development of disposable electrochemical magnetoimmunosensors for S. pneumoniae analysis as a rapid and sensitive method for future deployment for on-site diagnosis. A sandwich format was employed, where Protein A-coated MBs were used as a solid phase to immobilized, specific S. pneumoniae capture antibodies. The same antibody conjugated to an enzyme label, horseradish peroxidase (HRP), was employed as the detection antibody recognizing the captured S. pneumoniae cells. The electrochemical detection of the enzyme product was performed at a disposable gold screen-printed electrode (Au/SPE), using tetrathiafulvalene (TTF) as electron transfer mediator and H2O2 as the enzyme substrate. The immunosensor performance, including the selectivity against other streptococci, was evaluated, and the disposable magnetoimmunosensors were applied to the analysis of inoculated urine samples.
Section snippets
Apparatus and electrodes
Amperometric measurements were carried out with an ECO Chemie Autolab PSTAT 10 potentiostat using the software package GPES 4.9 (General Purpose Electrochemical System). A P-Selecta (Scharlab) ultrasonic bath, an autoclave (Raypa AES-75), a horizontal laminar flow Hood/Cabin Telstar mod. AH-100, an Optic Ivymen® System constant temperature incubator shaker (Comecta S.A.), a P-Selecta Agimatic magnetic stirrer, and a flow cytometer FC500 from Beckman Coulter, equipped with a 488 nm Ar-ion laser,
Results and discussion
The disposable magnetoimmunosensors developed are based on a sandwich configuration that is schematically displayed in Fig. 1. In brief, capture antibodies were coupled to the Protein A-coated MBs through the strong affinity of Protein A for the Fc part of IgG molecules. Then, the bacteria were captured by a 60-min conjugation step. This step was followed by a further 60-min conjugation of the HRP-labeled antibody to the bacteria-capture antibody-MB conjugate. After each step, MBs were
Conclusions
Disposable amperometric sensors, combining the use of modified MBs and specific capture and detection antibodies for the first time, have demonstrated a good performance for the rapid and selective quantification of S. pneumoniae. An acceptable detection limit, without pre-enrichment steps, of 3.0 × 103 cfu was achieved and up to 30 sensors per day can be prepared and used. It is important to remark that this is the first time, to our knowledge, that an immunosensor based on the use of novel
Acknowledgements
This research was supported by grants from the Dirección General de Investigación Científica y Técnica (SAF2006-00390 and SAF2009-10824). The financial support of Santander/Complutense Research Project PR 27/05-13953, the Spanish Ministerio de Ciencia e Innovación Research Project CTQ2009-09351BQU, and the AVANSENS Programme from the Comunidad de Madrid (S2009PPQ-1642) are also gratefully acknowledged. S. Campuzano acknowledges a “Juan de la Cierva” research contract to the Spanish Ministerio
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