Background and motivation
Nanochannels are showing to be an interesting platform for biosensing applications. This significant growth of interest in nanoporous materials has started from the platforms used for sensing designs with electrochemical detection, mimicking the processes that occur in natural ion channels and taking advantage of the inherent properties of the electrochemical techniques. Since maintaining a natural environment in an artificial device is a difficult task, the construction of fully and highly functional artificial systems with the ability of mimicking natural systems is demanded. This challenging subject led to the creation of different strategies to obtain artificial structures resembling the characteristics of ion channels commonly encountered in the membranes of living systems. These entire advantages make the nanochannel based biosensing systems a very promising research area that should bring wonderful scientific discoveries, with tremendous potential applications in fields such as diagnostics, safety-security and other industrial applications. Nanochannel based biosensing technologies would take advantages from the recent developments of nanoimprinting technologies (i.e. roll to roll printing) that together with other fabrication techniques and emerging nanomaterials may bring new opportunities in terms of robustness, mass production and better tailoring of nanochannel based platforms toward various applications better approaching this advantageous technology to more users interested in protein, DNA and cells sensing. [1,2]
Anodic aluminum oxide (AAO) based nanochannels as electrochemical sensing platforms
The properties of the anodized aluminum oxide (AAO) membranes are combined with the advantages of the screen-printed electrotransducers and the voltammetric detection mode. Due to this synergy simpler biosensing set-up alternatives and the corresponding methodologies for the detection of DNA  and proteins  even in real blood samples [5,6] have been developed. The detection principle is based on the monitoring of [Fe(CN)6]4- indicator ions diffusion through AAO membranes attached onto a screen-printed carbon electrotransducer by differential pulse voltammetry signal change after biomolecule recognition (Figure 1). This nanochannels based methodology has enormous potential applications, for example for the analysis of real samples, where the membrane can act at the same time as filter, minimizing matrix effects, and as a simple sensing platform (Figure 2).
Prussian blue nanoparticles (PBNPs) are also proposed as alternative red-ox indicators for the enhancement of the sensitivity of label-free immunoassays. The bigger size of the PBNPs compared with ionic indicators such as the [Fe(CN)6]4-/3- system and the consequent increase in the steric effects that hinder their diffusion to the electrodes is probably the main factor that allows to improve the detection limits of model human IgG from 200 µg mL-1 to 34 pg mL-1 levels (Figure 3). The optimized methodology is also applied for the detection of a very small protein, a cancer biomarker parathyroid hormone-related protein, PTHrP) spiked in HaCat cell culture medium at levels of 50 ng mL-1. 
Nanoparticles-based nanochannels assembled on flexible electrochemical sensing platforms
The practical application of nanoporous AAO membranes based electrochemical sensing systems is limited due to two main factors: i) the membrane and the electrotransducer have to be assembled prior the detection (not integrated system); and ii) the large thickness of AAO membranes (60 µm approx.) makes necessary the use of nanoparticle tags for the enhancement of nanochannel blockage. For these reasons, nanochannels with nanosized thickness integrated on the electrotransducer surface are highly required so as to overcome such limitations.
In the aforementioned context, we propose a novel, cheap, disposable and single-use assembled nanoparticles-based nanochannel platform onto a flexible substrate for label-free immunosensing (Figure 4). This sensing platform is formed by the dip-coating of a homogeneous and assembled monolayer of carboxylated polystyrene nanospheres (PS, 200 nm and 500 nm-sized) onto the working area of flexible screen-printed ITO/PET electrodes. The spaces between the self-assembled nanospheres generate well-ordered nanochannels, with inter-PS particles distances of around 65 and 24 nm respectively. The formed nanochannels are approached for the effectively immobilization of antibodies and subsequent protein detection based on the monitoring of [Fe(CN)6]4- flow through diffusion and the decrease in the differential pulse voltammetric (DPV) signal upon immunocomplex formation. The limit of detection obtained with such a label-free approach is lower when smaller is the PS diameter, and consequently the nanochannels size, reaching values as low as 580 ng/mL for human IgG, chosen as model protein. The system is highly specific against the main proteins present in physiological fluids and that could interfere in the detection of HIgG. Furthermore, low matrix effects are found when analysing a human urine sample, demonstrating the ability of the sensing system to perform analysis in real scenarios. 
1. Alfredo de la Escosura-Muñiz, Arben Merkoçi, “Nanochannels Preparation and Application in Biosensing”, ACS Nano 6 (9), 2012, 7556–7583.
2. Alfredo de la Escosura-Muñiz, Arben Merkoçi, “Nanochannels for electrical biosensing”, Trends in Analytical Chemistry, 79, 2016, 134-150.
3. Alfredo de la Escosura-Muñiz, Arben Merkoçi, “Nanoparticle based enhancement of electrochemical DNA hybridization signal using nanoporous electrodes”, Chem. Comm., 46(47), 2010, 9007-9009.
4. Alfredo de la Escosura-Muñiz, Arben Merkoçi, “Label-free voltammetric immunosensor using a nanoporous membrane based platform”, Electrochem. Commun. 12, 2010, 859-863.
5. A. De la Escosura-Muñiz, A. Merkoçi “A nanochannel / nanoparticle based filtering and sensing platform for direct detection of a cancer biomarker in blood”, Small, 7, 2011, 675-682. (Highlighted By Michael Berger. Copyright 2011 Nanowerk: http://www.nanowerk.com/spotlight/spotid=20257.php)
6. Alfredo de la Escosura-Muñiz, Wilanee Chunglok, Werasak Surareungchai, Arben Merkoçi. “Nanochannels for diagnostic of thrombin-related diseases in human blood”. Biosens. Bioelectron., 4, 2013, 24-31.
7. Marisol Espinoza-Castañeda, Alfredo de la Escosura-Muñiz, Alejandro Chamorro, Carmen de Torres, Arben Merkoçi, «Nanochannel array device operating through Prussian blue nanoparticles for sensitive label-free immunodetection of a Cancer biomarker«, Biosens. Bioelectron. 67, 2015, 107–114.
8. Alfredo de la Escosura-Muñiz, Marisol Espinoza-Castañeda, Madoka Hasegawa, Laetitia Philippe, Arben Merkoçi, «Nanoparticles-based nanochannels assembled on a plastic flexible substrate for label-free immunosensing«, Nano Research. 8(4), 2015, 1180–1188.