Ready-to-use protein G-conjugated gold nanorods for biosensing and biomedical applications.

BACKGROUND: Gold nanorods (GNRs) display unique capacity to absorb and scatter near infrared light, which arises from their peculiar composition of surface plasmon resonances. For this reason, GNRs have become an innovative material of great hope in nanomedicine, in particular for imaging and therapy of cancer, as well as in photonic sensing of biological agents and toxic compounds for e.g. biomedical diagnostics, forensic analysis and environmental monitoring. As the use of GNRs is becoming more and more popular, in all these contexts, there is emerging a latent need for simple and versatile protocols for their modification with targeting units that may convey high specificity for any analyte of interest of an end-user. RESULTS: We introduce protein G-coated GNRs as a versatile solution for the oriented immobilization of antibodies in a single step of mixing. We assess this strategy against more standard covalent binding of antibodies, in terms of biocompatibility and efficiency of molecular recognition in buffer, serum and plasma, in the context of the development of a direct immunoenzymatic assay. In both cases, we estimate an average of around 30 events of molecular recognition per particle. In addition, we disclose a convenient protocol to store these particles for months in a freezer, without any detrimental effect. CONCLUSIONS: The biocompatibility and efficiency of molecular recognition is similar in either case of GNRs that are modified with antibodies by covalent binding or oriented immobilization through protein G. However, protein G-coated GNRs are most attractive for an end-user, owing to their unique versatility and ease of bioconjugation with antibodies of her/his choice.

PAX2 gene mutations in pediatric and young adult transplant recipients: kidney and urinary tract malformations without ocular anomalies.

Heterozygous humans for PAX2 mutations show autosomal dominant papillorenal syndrome (PRS), consisting of ocular colobomas, renal hypo/dysplasia and progressive renal failure in childhood. PAX2 mutations have also been identified in patients with isolated renal hypo/dysplasia. Twenty unrelated children and young adults with kidney and urinary tract malformations and no ocular abnormalities were retrospectively recruited for PAX2 mutational analysis. All patients had undergone renal transplantation after end-stage renal disease. We identified two new sequence variations: (i) a deletion causing a frameshift (c.69delC) and (ii) a nucleotide substitution determining a splice site mutation (c.410+5 G/A) by predictive analysis. Therefore, we suggest PAX2 molecular analysis to be extended to all patients with congenital malformations of kidney and urinary tract (CAKUT).

Different approaches for the detection of thrombin by an electrochemical aptamer-based assay coupled to magnetic beads.

Different assay formats based on the coupling of magnetic beads with electrochemical transduction were compared here for the detection of thrombin by using a thrombin specific aptamer. By using the thrombin-binding aptamer, a direct and an indirect competitive assay for thrombin have been developed by immobilising the aptamer or the protein, respectively. Moreover, another strategy was based on the direct measurement of the enzymatic product of thrombin captured by the immobilised aptamer. All the assays were developed by coupling the electrochemical transduction with the innovative and advantageous use of magnetic beads. The assays based on the immobilisation of the protein were not successful since no binding was recorded between thrombin and its aptamer. With the direct competitive assay, when the aptamer was immobilised onto the magnetic beads, a detection limit of 430nM for thrombin was achieved. A lower detection limit for the protein (175nM) was instead obtained by detecting the product of the enzymatic reaction catalysed by thrombin. All these assays were finally compared with a sandwich assay which reached a detection limit of 0.45nM of thrombin demonstrating the best analytical performances. With this comparison the importance of a deep study on the different analytical approaches for thrombin detection to reach the performances of the best assay configuration has been demonstrated.

Development of an optical RNA-based aptasensor for C-reactive protein.

The development of a RNA-aptamer-based optical biosensor (aptasensor) for C-reactive protein (CRP) is reported. CRP is an important clinical biomarker; it was the first acute-phase protein to be discovered (1930) and is a sensitive systemic marker of inflammation and tissue damage. It has also a prognostic value for patients with acute coronary syndrome. The average concentration of CRP in serum is 0.8 ppm and it increases in response to a variety of inflammatory stimuli, such as trauma, tissue necrosis, infection and myocardial infarction. The interaction between the 44-base RNA aptamer and the target analyte CRP is studied. In particular, the influence of the aptamer immobilization procedure (chemistry, length, concentration), as well as the binding conditions, i.e., the influence on the binding of different buffers, the presence of Ca2+ ion and the specificity (against human serum albumin) have been evaluated. Using the best working conditions, we achieved a detection limit of 0.005 ppm, with good selectivity towards human serum albumin. Some preliminary experiments in serum are reported.

Polychlorinated biphenyls (PCBs) detection in milk samples by an electrochemical magneto-immunosensor (EMI) coupled to solid-phase extraction (SPE) and disposable low-density arrays.

An electrochemical immunosensor for polychlorinated biphenyl (PCB) detection based on graphite screen-printed low-density arrays and on magnetic beads is reported. The immunological reaction for the detection of PCBs is based on a direct competitive assay using alkaline phosphatase (AP) as enzymatic label. After the immunochemical recognition, the modified magnetic beads are captured by a magnet on the surface of the graphite working electrode. The electrochemical detection is thus achieved through the addition of the AP substrate (alpha-naphthyl-phosphate). Two different antibodies (sIgG anti-PCB28 and rIgG anti-PCB77) were tested and compared in terms of sensitivity and ability to recognise different congeners. The developed electrochemical magneto-immunosensor (EMI) was successfully combined with solid-phase extraction (SPE) for the analysis of PCBs in milk samples. In spiked samples a recovery of 80% was obtained. The proposed strategy offers great promise for rapid, simple, cost-effective, and on-site analysis of clinical, food and environmental samples, considering also that low-density arrays allow the simultaneous analysis of different processed samples.

Improvement of analytical performances of a disposable electrochemical immunosensor by using magnetic beads.

A comparison of two electrochemical immunosensing strategies for PCBs detection, based on the use of two different solid phases, is here discussed. In both cases, carbon-based screen-printed electrodes (SPEs) are used as transducers in a direct competitive immunoassay scheme, where PCBs in solution compete with the tracer PCB28-alkaline phosphatase (AP) labeled for antibodies immobilized onto the solid-phase. In the standard format (called EI strategy), SPEs are both the solid-phase for immunoassay and electrochemical transducers: in this case the immunochemical reaction occurs onto the working electrode. Finally, the enzymatic substrate is added and an electroactive product is generated and detected by electrochemical measurement. In order to improve the performances of the system, a new approach (called EMI strategy) is developed by using functionalized magnetic beads as solid phase for the competitive assay; only after the immunosensing step they are captured by a magnet onto the working surface of the SPE for the electrochemical detection. Experimental results evidenced that the configuration based on the use of separate surfaces for immunoassay and for electrochemical detection gave the best results in terms of sensitivity and speed of the analysis. The improvement of analytical performances of the immunosensor based on EMI strategy was also demonstrated by the analysis of some spiked samples.

Clinical and molecular markers of chronic interstitial nephropathy in congenital unilateral ureteropelvic junction obstruction.

PURPOSE: We evaluated clinical and biological variables, and their meaning as reliable markers of chronic interstitial nephropathy in a selected group of children with prenatally detected hydronephrosis who underwent pyeloplasty because of congenital unilateral ureteropelvic junction obstruction. MATERIALS AND METHODS: We reviewed the clinical, prenatal and postnatal ultrasonographic, and scintigraphic records of children for whom intraoperative biopsy records were available. We performed histological analysis, and evaluated tubulointerstitial immunostaining for vimentin and alpha-smooth muscle actin, and the immunohistochemical and mRNA expression of the renin-angiotensin system peptides and transforming growth factor-beta1. RESULTS: The children were divided in 2 groups according to the absence (group 1) or presence (group 2) of chronic interstitial nephropathy in the biopsy. Patients in group 2 were significantly younger at prenatal diagnosis (p = 0.031), and had decreased split renal function (p = 0.005) and worse drainage (p = 0.035) on preoperative diuretic renography. No differences were found in terms of degree of hydronephrosis, or its prenatal and postnatal variation. Group 2 biopsies exhibited greater immunostaining for alpha-smooth muscle actin and vimentin (p = 0.004 and p = 0.047, respectively), and transforming growth factor-beta1 mRNA levels (p = 0.06). Vimentin and alpha-smooth muscle actin positivity correlated with renin, angiotensin II receptors 1 and 2, and transforming growth factor-beta1 mRNA levels, and all correlated with preoperative split renal function and post-void washout. CONCLUSIONS: In congenital unilateral ureteropelvic junction obstruction chronic interstitial nephropathy and poor postoperative recovery seem to be associated with an earlier diagnosis of hydronephrosis, functional loss greater than 10% and worse scintigraphic drainage. Moreover, there is a strong correlation between molecular fibrogenic markers and histologically and scintigraphically demonstrated renal damage.